Formulations of recombinant furin

ABSTRACT

The present application provides stabilized formulations of furin (e.g., rfurin) containing a sugar, sugar alcohol, and/or non-ionic surfactant. As compared to non-stabilized compositions, the furin formulations disclosed herein retain greater amounts of furin activity and monomeric furin content, while reducing furin aggregation when stored and/or subjected to mechanical stress. Also provided are methods for stably diluting furin (e.g., rfurin) compositions.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application No. 61/492,712, filed Jun. 2, 2011, the content of which is expressly incorporated herein by reference in its entirety for all purposes.

FIELD OF THE APPLICATION

This application generally relates to the field of protein formulations.

BACKGROUND OF THE INVENTION

The family of the proteolytic mammalian subtilisin-like proprotein convertases (SPC or PC) is homologous with bacterial subtilisins and yeast Kex2p. To date seven distinct members of the SPC family have been identified, including furin, PC1 (also known as PC3), PC2, PACE4, PC4, PC5 (also known as PC6), PC7 (LPC, PC8, or SPC7), each of which exhibits unique tissue distribution.

Furin, also termed PACE (paired basic amino acid cleavage enzyme) is ubiquitously expressed in all mammalian tissues and cell lines and is capable of processing a wide range of bioactive precursor proteins in the secretory pathway, including also hormones, growth factors, receptors, viral and bacterial proteins, and plasma proteins. It is a calcium-dependent serine endoprotease structurally arranged into several domains, namely a signal peptide, propeptide, catalytic domain, middle domain, (also termed homo-B or P-domain), the C-terminally located cysteine-rich domain, transmembrane domain and the cytoplasmic tail. The furin protease cleavage site comprises a recognition sequence which is characterized by the amino acid sequence Arg-X-Lys/Arg-Arg (Hosaka et al., J Biol Chem. 1991; 266:12127-30).

Furin belongs to the family of the pro-protein convertases and is dependent on calcium (Ca²⁺). Furin specifically cleaves the C-terminal peptide bond of arginine within a specific sequence, containing arginine at positions −1 and −4. This sequence can be found in numerous human proteins, showing that furin plays a major role in the maturation of a number of human pro-proteins. Accordingly, furin is a proprotein convertase that processes latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that cleaves precursor proteins at their paired basic amino acid processing sites. One substrate for furin is von Willebrand Factor (vWF).

Furin (e.g., rfurin) is capable of converting pro-VWF (pro-von Willebrand factor) into mature VWF by cleaving the Arg741-Ser742 peptide bond of VWF. This maturation step is part of, for example, a rVWF production process which provides a therapy for von Willebrand Disease Type B. The production of activated recombinant proteins is of high clinical and diagnostic importance. For example, active or mature proteins, like mature VWF, may be used to control blood coagulation.

Furin formulations, particularly recombinant furin (rfurin) formulations, often have low shelf life (less than 6 months) due to a loss of rfurin activity from protein degradation (often from self-cleavage of the furin protein). In addition, when used in certain production processes, such as production of mature vWF, rfurin formulations are diluted, often by 200-fold, and the diluted rfurin generally shows a disproportionate loss of activity. Accordingly, there is a need in the field for methods and formulations that stabilize furin (e.g., rfurin) compositions, as well as methods for diluting concentrated furin (e.g., rfurin) compositions without losing a substantial fraction of furin activity.

BRIEF SUMMARY OF THE INVENTION

The present disclosure fulfills these and other needs in the field of furin (e.g., rfurin) formulation, storage, and manipulation, by providing methods and formulations for stabilizing furin (e.g., rfurin compositions). These needs are met, at least in part, by the discovery that the addition of sugars, sugar alcohols, and/or non-ionic surfactants significantly increases the stability of furin (e.g., rfurin) compositions during storage and mechanical stress.

Accordingly, the present invention provides highly stabilized furin formulations, particularly highly stabilized rfurin formulations that show increased stability under conditions of high temperature, agitation, and varying pH as compared to the starting (control) furin formulations.

In one aspect, the present disclosure provides a stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: from 8,000 U/mL to 500,000 U/mL rfurin; from 100 mM to 300 mM of a pharmaceutically acceptable salt; from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol; from 10 to 200 ppm non-ionic surfactant; from 10 to 200 mM buffering agent; and a pH from 5.5 to 7.5.

In one embodiment, the compositions provided above include 150 mM to 250 mM of a pharmaceutically acceptable salt. In another embodiment, the compositions provided above include 190±10 mM of a pharmaceutically acceptable salt. In another embodiment, the compositions provided above include 190 mM of a pharmaceutically acceptable salt.

In one embodiment of the compositions provided above, the pharmaceutically acceptable salt is sodium chloride. In another embodiment of the compositions provided above, the pharmaceutically acceptable salt is potassium chloride.

In one embodiment, the compositions provided above include 0.9±0.2 mM calcium. In another embodiment, the compositions provided above include 0.92 mM calcium.

In one embodiment, the compositions provided above include from 5% to 15% sugar or sugar alcohol. In another embodiment, the compositions provided above include 10±2% sugar or sugar alcohol. In another embodiment, the compositions provided above include 10% sugar or sugar alcohol.

In one embodiment of the compositions provided above, the sugar or sugar alcohol is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof. In another embodiment of the compositions provided above, the sugar or sugar alcohol is trehalose. In another embodiment of the compositions provided above, the sugar or sugar alcohol is sucrose.

In one embodiment, the compositions provided above include from 10 ppm to 100 ppm non-ionic surfactant. In another embodiment, the compositions provided above include from 50 ppm to 100 ppm non-ionic surfactant. In another embodiment, the compositions provided above include 75 ppm non-ionic surfactant.

In one embodiment of the compositions provided above, the non-ionic surfactant is polysorbate-80.

In one embodiment, the compositions provided above include from 50 mM to 150 mM buffering agent. In another embodiment, the compositions provided above include 90±10 mM buffering agent. In another embodiment, the compositions provided above include 91 mM buffering agent.

In one embodiment of the compositions provided above, the buffering agent comprises acetate. In another embodiment of the compositions provided above, the buffering agent comprises HEPES. In another embodiment of the compositions provided above, the buffering agent comprises MES. In another embodiment of the compositions provided above, the buffering agent comprises acetate and HEPES.

In one embodiment, the compositions provided above include from 25 mM to 75 mM acetate and from 25 to 75 mM HEPES. In another embodiment, the compositions provided above include 45±5 mM acetate and 45±5 mM HEPES. In another embodiment, the compositions provided above include 45 mM acetate and 46 mM HEPES.

In one embodiment, the compositions provided above have a pH from 5.5 to 7.0. In another embodiment, the compositions provided above have a pH from 5.5 to 6.5. In another embodiment, the compositions provided above have a pH of 6.0±0.2. In another embodiment, the compositions provided above have a pH of 6.0.

In one embodiment of the compositions provided above, the composition has increased stability when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.

In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin activity when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.

In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin monomer content when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.

In one embodiment of the compositions provided above, the composition has increased stability when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.

In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin activity when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.

In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin monomer content when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.

In one embodiment of the compositions provided above, the composition has increased stability when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.

In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin activity when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.

In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin monomer content when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.

In one aspect, the present disclosure provides a stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: from 8,000 U/mL to 500,000 U/mL rfurin; 190 mM sodium chloride; 0.92 mM calcium; 10% trehalose; 75 ppm polysorbate 80; 45 mM acetic acid; 46 mM HEPES; and a pH from 5.5 to 7.5.

In one embodiment, the compositions provided above have a pH from 5.5 to 7.0. In another embodiment, the compositions provided above have a pH from 5.5 to 6.5. In another embodiment, the compositions provided above have a pH of 6.0±0.2. In another embodiment, the compositions provided above have a pH of 6.0.

In one aspect, the present disclosure provides a method for diluting an aqueous rfurin composition, the method comprising adding a dilution buffer comprising from 10 ppm to 200 ppm of a non-ionic surfactant to the aqueous rfurin composition, thereby forming a diluted rfurin composition.

In one embodiment of the methods provided above, the dilution buffer comprises from 10 ppm to 100 ppm of a non-ionic surfactant. In another embodiment of the methods provided above, the dilution buffer comprises from 50 ppm to 100 ppm of a non-ionic surfactant. In another embodiment of the methods provided above, the dilution buffer comprises 75 ppm of a non-ionic surfactant.

In one embodiment of the methods provided above, the dilution buffer has a pH of from 5.5 to 7.5. In another embodiment of the methods provided above, the dilution buffer has a pH of from 5.5 to 6.5. In another embodiment of the methods provided above, the dilution buffer has a pH of 6.0±0.2. In another embodiment of the methods provided above, the dilution buffer has a pH of 6.0.

In one embodiment of the methods provided above, the diluted rfurin composition comprises at least 50% of rfurin activity present in the aqueous rfurin composition prior to dilution.

In one embodiment of the methods provided above, the diluted rfurin composition comprises at least 75% of rfurin activity present in the aqueous rfurin composition prior to dilution.

In one aspect, the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin), said formulation comprising: 8,000-52,000 U/mL rfurin; 1 mM to 300 mM of a pharmaceutically acceptable salt; 0.1 mM to 2 mM calcium chloride; 1% to 20% sugar and/or sugar dihydrate; 50 to 100 ppm of a surfactant; and 1 mM to 100 mM of a carboxylic acid, a buffering agent in sufficient concentration for maintaining a pH of 4.0-7.0, or both said carboxylic acid and said buffering agent.

In one embodiment, the formulations provided above include 180-200 mM salt.

In one embodiment of the formulations provided above, the salt is sodium chloride.

In one embodiment, the formulations provided above include 0.5-1.0 mM calcium.

In one embodiment, the formulations provided above include 5-15% sugar or sugar dihydrate.

In one embodiment of the formulations provided above, the sugar or sugar dihydrate is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof.

In one embodiment of the formulations provided above, the surfactant is polysorbate 80.

In one embodiment of the formulations provided above, the surfactant is present at a concentration of about 65 to about 85 ppm.

In one embodiment of the formulations provided above, the carboxylic acid is acetic acid.

In one embodiment of the formulations provided above, the carboxylic acid is in a concentration of 35-55 mM.

In one embodiment of the formulations provided above, the buffering agent is HEPES.

In one embodiment of the formulations provided above, the buffering agent is in a concentration of about 20 to about 100 mM.

In one embodiment of the formulations provided above, the formulation shows improved stability after storage at 37° C. as compared to a formulation that does not contain a sugar or sugar dihydrate.

In one embodiment of the formulations provided above, the formulation is prepared by adding rfurin to a composition comprising 1% polysorbate 80, 500 mM HEPES, 400 mM acetic acid, 1 mM calcium chloride, and trehalose dihydrate powder, wherein said composition has a pH of 6.0 prior to addition to said rfurin.

In one aspect, the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin), said formulation comprising: 8,000-57,000 U/mL rfurin; 190 mM sodium chloride; 0.92 mM calcium chloride; 10% w/w trehalose dihydrate; 75 ppm polysorbate 80; 45 mM acetic acid; and 46 mM HEPES.

In one aspect, the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin) that includes: (a) 8,000-52,000 U/mL rfurin; (b) 1 mM to 300 mM of a pharmaceutically acceptable salt; (c) 0.1 mM to 2 mM calcium chloride; (d) 1% to 20% sugar and/or sugar dihydrate; (e) 50 to 100 ppm of a surfactant; and (f) 1 mM to 100 mM of a carboxylic acid or a buffering agent in sufficient concentration for maintaining a pH of 4.0-7.0 or both the carboxylic acid and the buffering agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. rFurin in the control formulation—a temperature study analyzed using the furin activity assay. rFurin samples in the control formulation were incubated at: room temperature, 37° C., or 45° C. for up to seven days. The activities of rfurin samples are plotted against the number of days of incubation. Error bars are ±1 standard deviation; n=4.

FIG. 2. rFurin in the control formulation—a temperature study analyzed using SEC. The same samples as in FIG. 1 were analyzed using SEC. Relative rfurin peak heights are plotted against the number of days of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.

FIG. 3. rFurin in the control formulation incubated at 37° C. analyzed using SEC. rFurin samples in the control formulation were incubated at 37° C. for seven days. FIG. 3 shows an overlay of SEC absorption profiles at 280 nm of six time points. The same study as in FIG. 1.

FIG. 4. rFurin in the control formulation incubated at room temperature and 37° C. analyzed using Western Blotting. The same study as in FIG. 1. Lane: (1) MWM marker; (2) T=0; (3) rfurin incubated at room temperature for 1 day; (4) rfurin incubated at room temperature for 2 days; (5) rfurin incubated at room temperature for 3 days; (6) rfurin incubated at room temperature for 7 days; (7) MWM marker; (8) rfurin incubated at 37° C. for 1 day; (9) rfurin incubated at 37° C. for 2 days; (10) rfurin incubated at 37° C. for 3 days; and (11) rfurin incubated at 37° C. for 7 days.

FIG. 5. rFurin in the control formulation—a freeze/thaw study analyzed using the furin activity assay. rFurin activity is plotted against the number of freeze/thaw cycles. Error bars are ±1 standard deviation; n=4.

FIG. 6. rFurin in the control formulation—a freeze/thaw study analyzed using SEC. rFurin peak heights are plotted against the number of freeze/thaw cycles.

FIG. 7. rFurin in the control formulation—an agitation study analyzed using the activity assay. rFurin activity is plotted against the time of agitation. Error bars are ±1 standard deviation; n=4.

FIG. 8. rFurin in the control formulation—an agitation study analyzed using SEC. rFurin peak heights are plotted against the time of agitation.

FIG. 9. Comparison of different lots of rfurin using Western Blotting. Various lots of rfurin were analyzed using Western Blotting. Production order is denoted alphabetically. Lane: (1) MWM marker; (2) furin reference; (3) 03-04-09 (lot 018342, 2 mL vial); (4) 03-08-09 (lot 018365, 2 mL vial); (5) 03-15-09 (lot 018366, 2 mL vial); (6) 03-01-09 (lot 018315, 2 mL vial); (7) 07-26-10 (lot X572, 200 mL bottle); (8) 03-06-09 (lot 018365, 200 mL bottle); (9) 08-01-10 (lot X576, 200 mL bottle); (10) 03-09-09 (lot 018366, 200 mL bottle); (11) MWM marker; (12) furin reference; (13) 08-03-10 (lot X577, 200 mL bottle); (14) 07-24-10 (lot X573, 200 mL bottle); (15) 07-30-10 (lot X575, 200 mL bottle); (16) 018346 (QC Stability at −80° C.); (17) 018346 (QC Stability at −25° C.); (18) 018315 (QC Stability at −80° C.); and (19) 018315 (QC Stability at −25° C.).

FIG. 10. Impact of thawing method on rfurin activity analyzed using the furin activity assay. rFurin sample activities are shown as bars for various lots of rfurin thawed at either: 4° C. (left) or room temperature (RT; right). Error bars are ±1 standard deviation; n=4.

FIG. 11. Effect of pH 5 and 5.5 on rfurin activity analyzed using the furin activity assay. rFurin samples were incubated at 37° C. at either pH: 5.0, 5.5, or 6.0 (control formulation) for up to seven days. The activities of rfurin samples are plotted against the number of days of incubation. Error bars are ±1 standard deviation; n=4.

FIG. 12. Effect of pH 5 and 5.5 on rfurin activity analyzed using SEC. rFurin samples were incubated at 37° C. at either pH: 5.0, 5.5 or 6.0 (control formulation). Relative peak heights of rfurin are plotted against the number of days of incubation. The relative peak heights were calculated as the percentage of the rfurin peak height at time zero.

FIG. 13. rFurin stability at 37° C. in the control formulation analyzed using SEC. A rfurin sample in the control formulation was incubated for four days at 37° C. The overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.

FIG. 14. rFurin stability in MES pH 6.0 analyzed using SEC. A rfurin sample was spiked with MES (final C=100 mM, pH 6.0) and incubated for four days at 37° C. An overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.

FIG. 15. rFurin stability in HEPES pH 7.0 analyzed using SEC. A rfurin sample was spiked with HEPES (final C=100 mM, pH 7.0) and incubated for four days at 37° C. The overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.

FIG. 16. rFurin stability in HEPES pH 8.0 analyzed using SEC. A rfurin sample was spiked with HEPES (final C=100 mM, pH 8.0) and incubated for four days at 37° C. The overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.

FIG. 17. Effects of sucrose and polysorbate 80 additives on rfurin stability at 37° C. analyzed using SEC. The samples were incubated at 37° C. for four days. Relative peak heights of rfurin are plotted against the time of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.

FIG. 18. Effects of sucrose and polysorbate 80 on rfurin stability in the control formulation at 37° C. analyzed using the furin activity assay. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: control formulation without additives, control formulation plus 10% sucrose, and control formulation plus 10% sucrose plus 25 ppm polysorbate 80. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation=4.

FIG. 19. Effects of sucrose and polysorbate 80 on rfurin stability in MES buffer at 37° C. analyzed using the furin activity assay. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: control formulation without additives, spiked with MES (final C=100 mM, pH 6.0), spiked with MES (final C=100 mM, pH 6. plus 10% sucrose, and spiked with MES (final C=100 mM, pH 6.0) plus 10% sucrose plus 25 ppm polysorbate 80. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation; n=4.

FIG. 20. Comparison of rfurin stability at 37° C. in the control formulation to the formulation in MES buffer analyzed using the furin activity assay. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: control formulation without additives, control formulation plus 10% sucrose, and spiked with MES (final C=100 mM, pH 6.0) plus 10% sucrose. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation; n=4.

FIG. 21. Comparison of rfurin stability at 37° C. in sucrose to mannitol analyzed using the furin activity assay. All samples were spiked with HEPES/acetic acid, pH 6.0. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: no additives, 10% sucrose, 10% mannitol, 10% sucrose plus 25 ppm of polysorbate 80, and 10% mannitol plus 25 ppm of polysorbate 80. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation; n=4.

FIG. 22. rFurin Stability at 37° C.: sucrose vs. mannitol analyzed using SEC. The same samples as in FIG. 21 were analyzed using SEC. Relative rfurin peak heights are plotted against the time of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.

FIG. 23. rFurin stability in trehalose after 5 days of incubation at 37° C. analyzed by the furin activity assay. The effects of varied amounts of trehalose and polysorbate 80 on rfurin stability at 37° C. are examined using the furin activity assay. First, all samples were spiked with HEPES and acetic acid (final concentrations: HEPES—50 mM, acetic acid—50 mM, pH 6.0) then, either 10% sucrose or various amounts of trehalose and/or polysorbate 80 were added. A rfurin sample in the control formulation is also included. The data are sorted from the highest to lowest activity values after 5 days of incubation at 37° C. (right bar in each pair). The left bar in each pair depicts furin activity values at time zero. Error bars are ±1 standard deviation; n=4.

FIG. 24. rFurin activity in trehalose after 5 days of incubation at 37° C. The same samples as in FIG. 23 are displayed after recalculation to show what percentage of each sample's initial activity remained after five days of incubation at 37° C. A rfurin sample in the control formulation is also included. Error bars are ±1 standard deviation; n=4.

FIG. 25. rFurin stability in trehalose after 5 days of incubation at 37° C. analyzed using SEC. The same samples as in FIG. 24. The graph shows what percentage of each sample's initial rfurin peak remained after five days of incubation at 37° C. A rfurin sample in the control formulation is also included.

FIG. 26. rFurin stability in trehalose after 5 days of incubation at 35° C. analyzed using Western Blotting. The same samples as in FIG. 24. The samples are paired: first sample—time zero, second sample—incubated at 37° C. for 5 days. Lanes: (1) MWM marker; (2) furin reference, (3) control; (4) control formulation, T=0; (5) control formulation, 5 days; (6) 10% sucrose, T=0; (7) 10% sucrose, 5 days; (8) 2% trehalose, T=0; (9) 2% trehalose, 5 days; (10) 10% trehalose, T=0; (11) 10% trehalose, 5 days; (12) MWM marker; (13) furin reference; (14) control; (15) 5% trehalose, T=0; (16) 5% trehalose, 5 days; (17) 5% trehalose, +10 ppm Tween80, T=0; (18) 5% trehalose, +10 ppm Tween80, 5 days; (19) 5% trehalose, +25 ppm Tween80, T=0; (20) 5% trehalose, +25 ppm Tween80, 5 days; (21) 5% trehalose, +100 ppm Tween80, T=0; and (22) 5% trehalose, +100 ppm Tween80, 5 days.

FIG. 27. Effect of the trehalose content on rfurin activity tested after 5 days of incubation at 37° C. The same experiment as in FIG. 24. First, all samples were spiked with HEPES and acetic acid (final concentrations: HEPES—50 mM, acetic acid—50 mM, pH 6.0) then, various amounts of trehalose were added. furin activity after five days of incubation at 37° C. is plotted against the percentage of trehalose added to the samples. Error bars are ±1 standard deviation; n=4.

FIG. 28. Effect of the trehalose content on rfurin stability at 37° C. analyzed using SEC after 5 days of incubation. The same experiment as in FIG. 27. rFurin peak heights after five days of incubation at 37° C. are plotted against the percentage of trehalose added to the samples.

FIG. 29. Effect of polysorbate 80 content on rfurin activity tested after 5 days of incubation at 37° C. The same experiment as in FIG. 24. First, all samples were spiked with HEPES, acetic acid (final concentrations: HEPES—50 mM, acetic acid—50 mM, pH 6.0), and 5% trehalose then, various amounts of polysorbate 80 were added. furin activity after five days of incubation at 37° C. is plotted against the amount of polysorbate 80 added to the samples. Error bars are ±1 standard deviation; n=4.

FIG. 30. Effect of Polysorbate 80 content on rfurin stability at 37° C. analyzed using SEC after 5 days of incubation. The same samples as in FIG. 29. rFurin peak heights after five days of incubation at 37° C. are plotted against the amount of polysorbate 80 added to the samples.

FIG. 31. rFurin in the highly stabilized formulation—agitation study analyzed using the furin activity assay. The rfurin samples in the highly stabilized formulation were spiked with various amount of polysorbate 80. The graph shows furin activity values before and after three hours of agitation. The data are sorted from the highest to the lowest activity. Error bars are ±1 standard deviation; n=4. Left bars in each pair show activity at time 0, right bars in each pair show activity after agitation.

FIG. 32. rFurin in the highly stabilized formulation—agitation study analyzed using SEC. The same experiment as in FIG. 31. The graph shows rfurin peak heights before and after three hours of agitation. The data are sorted from the highest to the lowest. Left bars in each pair show activity at time 0, right bars in each pair show activity after agitation.

FIG. 33. rFurin in the highly stabilized formulation—effects of polysorbate content in an agitation study analyzed using the furin activity assay. The same samples as in FIG. 32. furin activity after three hours of agitation is plotted against the content of polysorbate 80. Error bars are ±1 standard deviation; n=4.

FIG. 34. rFurin in the highly stabilized formulation—effect of polysorbate 80 in agitation study analyzed using SEC. The same samples as in FIG. 32. rFurin peak heights after three hours of agitation are plotted against the content of polysorbate 80.

FIG. 35. rFurin in the control formulation—agitation study analyzed using SEC. The chromatogram shows an overlay of SEC absorption profiles at 280 nm comparing rfurin samples in the control formulation before and after three hours of agitation. The rfurin peak of the agitated sample is significantly smaller than the control sample. The absence of high molecular mass peaks in the agitated sample is observed (see paragraph 3.5.1 for explanation).

FIG. 36. UV spectra of rfurin in the control formulation (the same samples as in FIG. 35). The UV spectrum of the rfurin sample after three hours of agitation shows a slanted and extremely elevated profile compared to the sample before agitation, which indicates the presence of a significant level of aggregates in the agitated sample.

FIG. 37. UV spectra of the rfurin samples in the highly stabilized formulation without polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows an elevated profile compared to the sample before agitation, which indicates the presence of significant level of aggregates in the agitated sample.

FIG. 38. rFurin in the highly stabilized Formulation without polysorbate 80—an agitation study analyzed using SEC. The chromatogram shows an overlay of SEC absorption profiles at 280 nm comparing rfurin samples in the highly stabilized formulation without polysorbate 80 before and after three hours of agitation. The rfurin peak of the agitated sample is significantly smaller than the control sample. The absence of high molecular mass peaks is observed.

FIG. 39. UV spectra of the rfurin samples in the highly stabilized formulation containing 10 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a slightly elevated profile compared to the sample before agitation, which indicates the presence of aggregates in the agitated sample. The ‘agitated’ spectra is closer to the ‘before agitated’ spectra, relative to those seen in samples without polysorbate (FIGS. 36 and 37), which indicated that that the level of aggregation is less than that seen in samples without polysorbate.

FIG. 40. UV spectra of the rfurin samples in the highly stabilized formulation containing 25 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a slightly elevated profile compared to the sample before agitation, which indicates presence of a small amount of aggregates in the agitated sample.

FIG. 41. UV spectra of the rfurin samples in the highly stabilized formulation containing 50 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a very similar profile to the sample before agitation. Neither spectra is slanted or elevated, which indicates that neither sample contains significant amounts of large aggregates.

FIG. 42. UV spectra of the rfurin samples in the highly stabilized formulation containing 100 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a very similar profile to the sample before agitation. Neither spectra is slanted or elevated, which indicates that neither sample contains significant amounts of large aggregates.

FIG. 43. rFurin in the highly stabilized Formulation—a freeze/thaw study analyzed using the furin activity assay. rFurin samples in the highly stabilized formulation were spiked with different amounts of polysorbate 80. The data show rfurin activity in the samples before and after 5 freeze/thaw cycles. The data are sorted from the highest to the lowest. Error bars are ±1 standard deviation; n=4.

FIG. 44. rFurin in the highly stabilized formulation—a freeze/thaw study analyzed using SEC. The same samples as in FIG. 43. The data show the rfurin peak heights before and after 5 freeze/thaw cycles. The data are sorted from the highest to the lowest.

FIG. 45. rFurin in the highly stabilized Formulation—a freeze/thaw study analyzed using SEC. The same samples as in FIG. 44. The relative peak heights of rfurin are plotted against the number of freeze/thaw cycles. The relative peak heights were calculated as the percentages of the rfurin peak heights before the first freeze/thaw cycle.

FIG. 46. rFurin stability in the highly stabilized formulation at 37° C. analyzed using the furin activity assay. rFurin samples in either the control formulation or in the highly stabilized formulation were incubated at 37° C. for 4 days. Error bars are ±1 standard deviation; n=4.

FIG. 47. rFurin stability in the highly stabilized formulation at 37° C. analyzed using the furin activity assay. The same samples as in FIG. 46 recalculated. The relative activity of each sample was calculated as the percentage of its initial activity. Error bars are ±1 standard deviation; n=4.

FIG. 48. rFurin stability in the highly stabilized formulation at 37° C. analyzed using SEC. The same samples as in FIG. 46. The relative peak height of rfurin for every sample was calculated as the percentage of its initial peak height. Error bars are ±1 standard deviation; n=4.

FIG. 49. Effect of temperature on the pH (B) and conductivity (A) of the highly stabilized formulation stock buffer (500 mM HEPES, 400 mM acetic acid, 1 mM CaCl₂, pH 6.0).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present disclosure is based in part on the discovery that aqueous furin compositions can be stabilized against various chemical and mechanical stresses by the addition of sugars, sugar alcohols, and non-ionic surfactants. The aqueous furin compositions described herein are significantly more stable when stored at or above room temperature, as well as when subjected to mechanical stress, as compared to similar furin compositions lacking sugars, sugar alcohols, and non-ionic surfactants. The present disclosure is also based in part on the discovery that the addition of non-ionic surfactants to furin dilution buffers enables a greater recovery of furin activity when the enzyme is diluted to lower concentrations used during the manufacture of various recombinant biologics, such as VWF.

Advantageously, the studies described herein demonstrate that the addition of sugar or sugar alcohols to a furin composition enhances the stability of the composition when stored over a period of time. For example, it is shown herein that the inclusion of as little as 2% sugar or sugar alcohol in an aqueous furin formulation can increase the stability of the composition by greater than 10%. It is further shown that inclusion of 10% sugar or sugar alcohol in an aqueous furin formulation can increase the stability of the composition by greater than 75%.

Advantageously, the studies described herein also demonstrate that the addition of non-ionic surfactant to a furin composition enhances the stability of the composition when subjected to mechanical stress. For example, it is shown herein that the inclusion of as little as 10 ppm of non-ionic surfactant in an aqueous furin formulation can increase the stability of the composition by greater than 75%.

Advantageously, the studies described herein also demonstrate that the inclusion of non-ionic surfactant to a furin dilution buffer increases the recovery of furin activity after dilution. For example, it is shown herein that the inclusion of 75 ppm of non-ionic surfactant in a furin dilution buffer increases the recovery of furin activity after dilution by 3- to 4-fold.

Accordingly, the present disclosure provides compositions of highly stabilized furin formulations. Although the majority of the discussion herein is in terms of highly stabilized formulations of recombinant furin (rfurin), it will be appreciated that any furin protein, including furin isolated from a subject or any derivatives or mutants of furin, can be included in the formulations of the present invention.

In one aspect, the present disclosure provides highly stabilized furin formulations that show improved stability over control formulations when assayed after subjecting the formulation to one or more stressors, including without limitation exposure of the formulations to a range of temperatures, to several freeze/thaw cycles, and/or to agitation. Improved stability is generally assessed by level of furin activity—for example, after storage at higher than ambient room temperature, a highly stabilized furin formulation will show a higher level of activity than a control formulation stored under identical conditions as compared to the activity of each prior to storage (i.e., at time=0). Methods for assessing the stability of a furin formulation are described herein.

In further aspects, the present disclosure provides methods for forming highly stabilized furin formulations of the present invention. Such methods include methods for stabilizing a formulation of furin for storage, including frozen storage, storage at ambient room temperature or above, or lyophilization.

In yet other aspects, the present disclosure provides methods for diluting concentrated furin solutions that result in increased retention of furin activity after dilution. As compared to furin compositions diluted with solutions lacking non-ionic surfactant, aqueous compositions of furin diluted according to the methods provided herein retain 3- to 4-times more enzymatic activity.

II. Definitions

As used herein, the term “furin” refers to any protein or polypeptide with furin activity, particularly the ability to cleave the peptide bond between residues Arg-763 and Ser-764 of the pro-von Willebrand Factor (pro-VWF) polypeptide. In an exemplary embodiment, furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to that of NP_(—)002560.1 (human furin preproprotein). In an exemplary embodiment, furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to amino acids 25-794 of NP_(—)002560.1 (human furin proprotein). In an exemplary embodiment, furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to amino acids 108-794 of NP_(—)002560.1 (human mature furin protein). As used herein, furin polypeptides also include natural variants of furin with VWF cleaving activity, as well as modified furin constructs with VWF cleaving activity. As used herein, furin encompasses any natural variants, alternative sequences, isoforms or mutant proteins that retain some basal activity (for example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more activity as compared to the activity of wild-type furin). Examples of furin mutations found in the human population include, without limitation, A43E, A43G, A43V, R50W, Y64C, L77P, R81C, E97A, E97V, V109M, V109L, R130W, A139V, P169T, N245S, E271K, Q339R, N407S, E457Q, R464W, K469R, S524Y, T536S, L570F, D624N, A642T, S685P, V728I, V735I, R745Q, P772L, and A793T. Furin polypeptides also include polypeptides containing post-translational modification. For example, it has been shown that furin is phosphorylated at residues S773 and S775 and predicted that furin is glycosylated at residues N387, N440, and N553.

In the context of the present disclosure, furin proteins include recombinant furin polypeptides as well as native furin polypeptides isolated from a source material (e.g., tissue or blood). Furin polypeptides (recombinant and source purified) may be derived from any suitable organism, for example, a mammal such as a primate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster, gerbil, canine, feline, and biologically active derivatives thereof. Mutant and variant furin polypeptides having VWF cleaving activity are also embraced, as are functional fragments, and fusion proteins comprising furin polypeptides. Furthermore, the furin polypeptides described herein may further comprise tags that facilitate purification, detection, or both. The furin polypeptides described herein may further be modified with a therapeutic moiety or a moiety suitable for imaging in vitro or in vivo.

Proteolytically active recombinant furin may be prepared by expression in cell culture (e.g., mammalian cell culture). Non-limiting examples of expression and purification methods for preparing recombinant furin are described in WO 1991/06314, WO 1992/09698, U.S. Pat. Nos. 6,210,929 and 6,596,526, as well as in U.S. Patent Application Publication Nos. 2009/0181423 and 2009/0304669, the contents of which are hereby incorporated by reference in their entireties for all purposes.

As used herein, “activity” refers to a functional activity or activities of furin or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, biological activity, including participation in the proteolytic maturation of proprotein substrates and cleavage of test substrates such as Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; AMC=7-amino-4-methoxy coumarin). Substrates for furin include von Willebrand factor, proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, and the beta subunit of pro-nerve growth factor. In one embodiment, one Unit (U) of furin activity is defined as the amount of furin that releases 1 pmol of AMC from Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1) per minute.

As used herein, the term “stability” (such as furin stability or furin formulation stability) is used in a structural context, e.g., relating to the structural integrity of a protein, or in a functional context, e.g., relating to a protein's ability to retain its function and/or activity over time. As will be appreciated, the protein under discussion may be contained within a formulation in accordance with the methods and compositions described herein, and the stability of that protein refers to its stability in that formulation. In one embodiment, the stability of a furin composition is determined by measuring the furin activity of the composition. For example, by using a detectable furin substrate such as Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; e.g., ALX-260-040-M001 sold by Enzo Life Sciences), for example, in an assay as described in Malloy SS, et al., J Biol Chem. 1992 Aug. 15; 267(23):16396-402, the content of which is hereby incorporated herein by reference in its entirety for all purposes. In one embodiment, the stability of furin composition formulated with sugar, sugar alcohol, and/or non-ionic surfactant, as described herein, is compared to a furin composition formulated without the sugar, sugar alcohol, and/or non-ionic surfactant.

As used herein, a “storage stable” aqueous furin composition refers to a furin polypeptide solution (e.g., a rfurin polypeptide solution) that has been formulated to increase the stability of the protein in solution, for example by at least 10%, over a given storage time. In the context of the present disclosure, a furin polypeptide solution (e.g., a rfurin polypeptide solution) can be made “storage stable” by the addition of a sugar, sugar alcohol, or non-ionic surfactant as a stabilizing agent. In some embodiments, the stability of the furin polypeptide in any given formulation can be measured, for example, by monitoring the formation of aggregates, loss of bulk enzymatic activity, or formation of degradation products, over a period of time. The absolute stability of a formulation, and the stabilizing effects of the sugar, sugar alcohol, or non-ionic surfactant, will vary dependent upon the particular composition being stabilized. In one embodiment, the stability of a furin composition is determined by measuring the furin activity of the composition. For example, by using a detectable furin substrate such as Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; e.g., ALX-260-040-M001 sold by Enzo Life Sciences), in an assay, for example, as described in Malloy SS, et al., J Biol Chem. 1992 Aug. 15; 267(23):16396-402, the content of which is hereby incorporated by reference in its entirety for all purposes. In one embodiment, the stability of furin composition formulated with sugar, sugar alcohol, and/or non-ionic surfactant, as described herein, is compared to a furin composition formulated without the sugar, sugar alcohol, and/or non-ionic surfactant.

As used herein, “shelf-life” refers to the period of time a formulation maintains a predetermined level of stability at a predetermined temperature. In particular embodiments, the predetermined temperature refers to frozen (e.g., −80° C., −25° C., 0° C.), refrigerated (e.g., 0° to 10° C.), or room temperature (e.g., 18° C. to 32° C.) storage.

As used herein, the term “time of stability” refers to the length of time a formulation is considered stable. For example, the time of stability for a formulation may refer to the length of time for which the level of protein aggregation and/or degradation in the formulation remains below a certain threshold (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.), and/or the length of time a formulation maintains biological activity above a certain threshold (e.g., 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, etc.) of the amount of activity present in the formulation at the start of the storage period.

In the context of the present disclosure, a storage stable aqueous composition of a furin polypeptide (e.g., rfurin polypeptide) formulated with a sugar, sugar alcohol, and/or non-ionic surfactant will have a longer time of stability than a composition of the same furin polypeptide formulated without the sugar, sugar alcohol, and/or non-ionic surfactant. In some embodiments, a storage stable aqueous composition of a furin polypeptide, will have a time of stability that is, for example, at least 10% greater than the time of stability for the same composition formulated in the absence of the sugar, sugar alcohol, and/or non-ionic surfactant, or at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% greater, or at least 2 times greater, or at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 9.5 times, 10 times, or more times greater than the time of stability for the same composition formulated in the absence of the sugar, sugar alcohol, and/or non-ionic surfactant.

As used herein, “storage” means that a formulation is not immediately administered to a subject or utilized in a production process once prepared, but is kept for a period of time under particular conditions (e.g. particular temperature) prior to use. For example, a furin formulation can be kept for days, weeks, months or years, prior to administration to a subject under varied temperatures such as frozen (e.g., −80° C., −25° C., 0° C.), refrigerated (e.g., 0° to 10° C.), or room temperature (e.g., 18° C. to 32° C.). As will be appreciated, such formulations may be liquid or lyophilized formulations.

As used herein, the term “about” denotes an approximate range of plus or minus 10% from a specified value. For instance, the language “about 20%” encompasses a range of 18-22%.

As used herein, when referring to a concentration of an individual component of a composition, the phrases “no more than X” and “from 0 to X” are equivalent and refer to any concentration between and including 0 and X. For example, the phrases “a concentration of no more than 2%” and “a concentration of from 0% to 2%” are equivalent and include 0%, 1%, and 2%.

As used herein, when referring to a concentration of an individual component of a composition, the phrases “no less than X” refers to any concentration X or higher. For example, the phrase “a concentration of no less than 98%” includes 98%, 99%, and 100%.

As used herein, when referring to a concentration of an individual component of a composition, the phrases “between X and Y” and “from X to X” are equivalent and refer to any concentration between and including X and Y. For example, the phrases “a concentration of between 49% and 51%” and “a concentration of from 49% to 51%” are equivalent and include 49%, 50%, and 51%.

As used herein, a “sugar” refers to monosaccharides having the general formula C_(x)H_(2y)O_(y) (linear) or C_(x)H_((2y-1))O_(y) (cyclic), and disaccharides consisting of two monosaccharide units formed through a dehydration reaction. Monosaccharides can be classified by the number of carbon atoms they contain: diose (2), triose (3), tetrose (4), pentose (5), hexose (6), heptose (7), etc. Accordingly, as used herein, a C(X) sugar refers to a sugar containing X-number of carbon molecules. For example, a C(5) sugar refers to a pentose sugar, while a C(6) sugar refers to a hexose sugar. Non-limiting examples of sugars that may be used in the formulations provided herein include: diose sugar glycolaldehyde, triose sugars glyceraldehyde and dihydroxyacetone; tetrose sugars erythrose, threose, and erythrulose; pentose sugars arabinose, lyxose, ribose, xylose, ribulose, and xylulose; hexose sugars allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose; heptose sugars sedoheptulose, mannoheptulose, and L-glycero-D-manno-heptose; and all possible combinations of disaccharide sugars formed thereform, including without limitation sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, and xylobiose.

As used herein, a “sugar alcohol” refers to a hydrogenated form of a mono- or disaccharide, whose carbonyl group has been reduced to form a primary or secondary hydroxyl. In one embodiment, the sugar alcohol has between about 4 and about 8 carbon atoms. Non-limiting examples of sugar alcohols that may be used in formulations provided herein include glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.

As used herein, a “pharmaceutically acceptable salt” refers to a salt that is safe for administration to a subject (e.g., a human) in a drug formulation. The selection and use of pharmaceutically acceptable salts is well known in the art, for example, see Stahl and Wermuth, Pharmaceutical Salts Properties, Selection, and Use, 2nd Revised edition, Wiley, Hoboken, N.J. In certain embodiments, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.

As used herein, the term “non-ionic surfactant” refers to a surface active agent that is non-ionized under physiologically relevant conditions. Non-limiting examples of non-ionic surfactants useful for the stabilized aqueous furin compositions provided herein include: non-ionic water soluble mono-, di-, and tri-glycerides (e.g., propylene glycol dicarpylate/dicaprate (e.g. MIGLYOL® 840), medium chain mono- and diglycerides (e.g. CAPMUL® and IMWITOR® 72), medium-chain triglycerides (e.g. caprylic and capric triglycerides such as LAVRAFAC, MIGLYOL® 810 or 812, CRODAMOL® GTCC-PN, and SOFTISON 378), long chain monoglycerides (e.g. glyceryl monooleates such as PECEOL®, and glyceryl monolinoleates such as MAISINE®), polyoxyl castor oil (e.g. macrogolglycerol ricinoleate, macrogolglycerol hydroxystearate, macrogol cetostearyl ether)); non-ionic water soluble mono- and di-fatty acid esters of polyethyelene glycol; non-ionic water soluble sorbitan fatty acid esters (e.g., sorbitan monolaurates such as polyoxyethylene (20) sorbitan monolaurate (TWEEN 20) and sorbitan monolaurate (SPAN 20); sorbitan monopalmitates such as polyoxyethylene (20) sorbitan monopalmitates (TWEEN 40) and sorbitan monopalmitate (SPAN 40); sorbitan monostearates such as polyoxyethylene (20) sorbitan monostearate (TWEEN 60) and sorbitan monostearate (SPAN 60); sorbitan monooleates such as polyoxyethylene (20) sorbitan monooleate (TWEEN 80) and sorbitan monooleate (SPAN 80); sorbitane trioleates such as sorbitane trioleate (SPAN 85); and sorbitan tristearates such as sorbitan tristearate (SPAN65)); polyglycolyzed glycerides (e.g., lauroyl macrogol-6 glycerides (Labrafil® M2130CS); d-α-tocopheryl polyethyleneglycol 1000 succinate (TPGS), poyethyleneglycol 660 12-hydroxystearate (SOLUTOL® HS 15), polyoxyl oleate and stearate (e.g. PEG 400 monostearate and PEG 1750 monostearate)); non-ionic water soluble triblock copolymers (e.g. poly(ethyleneoxide)/poly-(propyleneoxide)/poly(ethyleneoxide) triblock copolymers such as methyl-oxirane polymer with oxirane BHT (PLURONIC® F-127)).

In one embodiment, storage stable compositions of furin (e.g., rfurin) are provided which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof.

As used herein, the term “biologically active derivative,” when used in the context of furin polypeptide, also embraces polypeptides obtained via recombinant DNA technology. This may include any method known in the art for (i) the production of recombinant DNA by genetic engineering, e.g., via reverse transcription of RNA and/or amplification of DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, e.g., via electroporation or microinjection, (iii) cultivating said transformed cells, e.g., in a continuous or batch-wise manner, (iv) expressing furin protein, e.g., constitutively or upon induction, and (v) isolating said furin protein, e.g., from the culture medium or by harvesting the transformed cells, in order to (vi) obtain substantially purified recombinant furin protein, e.g., via ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and the like. The term “biologically active derivative” includes also chimeric molecules such as e.g., a furin protein, or functional fragment thereof, in combination with a second polypeptide, e.g., an immunoglobulin Fc domain or an albumin domain, in order to improve the biological/pharmacological properties such as e.g., half life of the furin protein in the circulation system of a mammal, particularly a human.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a buffering agent” refers to one agent or mixtures of such agents, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, compositions, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

As used herein, “BDS” refers to “Bulk Drug Substance.”

III. Stabilized Aqueous Compositions of Recombinant Furin

In one aspect, the present disclosure provides stabilized aqueous formulations of furin, e.g., rfurin. The following embodiments are based in part on the discovery that inclusion of a sugar, sugar alcohol, and/or non-ionic surfactant stabilizes aqueous furin compositions, as compared to compositions lacking the sugar, sugar alcohol, and/or non-ionic surfactant.

As will be recognized by one of skill in the art, furin compositions (e.g., rfurin compositions) formulated according to the embodiments provided herein may contain, in addition to the components explicitly disclosed, counter ions contributed by the inclusion of solution components or pH modifying agents, for example, sodium or potassium contributed from an acetate salt, sodium hydroxide, or potassium hydroxide or chloride contributed by calcium chloride or hydrochloric acid. In the context of the present disclosure, a storage stable furin composition (e.g., rfurin) consisting of or consisting essentially of a given formulation may further comprise one or more counter ion, as necessitated by the formulation process at a particular pH.

In one embodiment, a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at room temperature (i.e., between 18° C. and 32° C.) for a period of time. For example, in one embodiment, a storage stable, aqueous immunoglobulin composition will be stable when stored at room temperature for at least 4 days. In other embodiments, the composition will be stabile at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days. In other embodiments, the composition will be stable for at least 1 month. In yet other embodiments, the composition will be stable for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months. In certain embodiments, room temperature refers to between 20° C. and 30° C., between 21° C. and 29° C., between 22° C. and 28° C., between 23° C. and 27° C., between 24° C. and 26° C., or about 25° C. In a specific embodiment, the composition will be stable for an extended period of time when stored at a temperature between 20° C. and 25° C.

In one embodiment, a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at refrigerated temperature (i.e., between 2° C. and 10° C.) for a period of time. For example, in one embodiment, a storage stable, aqueous immunoglobulin composition will be stable when stored at refrigerated temperature for at least 4 days. In other embodiments, the composition will be stabile at refrigerated temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days. In other embodiments, the composition will be stable for at least 1 month. In yet other embodiments, the composition will be stable for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months. In a specific embodiment, the composition will be stable for an extended period of time when stored at a temperature between 2° C. and 8° C.

In one embodiment, a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at elevated temperature (i.e., between 32° C. and 42° C.) for a period of time. For example, in one embodiment, a storage stable, aqueous immunoglobulin composition will be stable when stored at elevated temperature for at least 4 days. In other embodiments, the composition will be stabile at elevated temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days. In other embodiments, the composition will be stable for at least 1 month. In yet other embodiments, the composition will be stable for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months. In a specific embodiment, the composition will be stable for an extended period of time when stored at a temperature between 35° C. and 40° C.

In one embodiment, a stored furin composition is considered storage stable as long as the composition maintains at least 40% of the furin activity present at the start of the storage period (e.g., at time=0). In another embodiment, a stored composition is considered stable as long as the composition maintains at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the furin activity present at the start of the storage period (e.g., at time=0). In one embodiment, furin activity is measures in an assay as described in Malloy S S, et al., J Biol Chem. 1992 Aug. 15; 267(23):16396-402.

In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% more furin activity after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage more furin activity after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.

In one embodiment, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 50%. In other embodiments, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.

In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% less furin present in an aggregated state after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage less furin present in an aggregated state after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.

In one embodiment, a stored furin composition is considered stable as long as the composition maintains at least 40% of the starting furin activity (e.g., at time=0) after being subjected to mechanical stress. In another embodiment, a stored composition is considered stable as long as the composition maintains 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the starting furin activity (e.g., at time=0) after being subjected to mechanical stress. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).

In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% more furin activity after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage more furin activity after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).

In one embodiment, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 50% after being subjected to mechanical stress. In other embodiments, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after being subjected to mechanical stress. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).

In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% less furin present in an aggregated state after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage less furin present in an aggregated state after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).

While the furin (e.g., rfurin) formulations described in this application can be lyophilized and reconstituted in the indicated concentrations, it will be appreciated that these preparations can also be reconstituted in more dilute form. For example, a preparation according the present disclosure which is lyophilized and/or normally reconstituted in 2 mL of solution can also be reconstituted in a larger volume of diluent, such as 5 mL. Likewise, lyophilized furin (e.g., rfurin) formulations can also be reconstituted in more concentrated form. For example, a preparation according the present disclosure which is lyophilized and/or normally reconstituted in 2 ml of solution can also be reconstituted in a smaller volume, such as 1 mL.

Advantageously, in one aspect, the highly stabilized furin (e.g., rfurin) formulations of the present invention are combined with a diluent that confers increased furin recovery when the resultant composition is used in a production method, for example, in the maturation of rVWF (also referred to herein as an “rVWF maturation method”). Maturation of pro-von Willebrand factor (vWF) to its active form requires proteolytic processing after a pair of dibasic amino acids (-Lys-Arg-) at residue 763. It has been shown that vWF is preferentially processed by the paired dibasic amino acid-cleaving enzyme furin. Production processes for vWF thus include the use of furin, preferably in a highly stabilized formulation. In a further aspect, the highly stabilized formulation in this diluent increases furin activity recovery in the rVWF maturation step by three to four times compared to control formulations placed in control diluents.

In certain aspects, the highly stabilized formulations of the invention have a shelf life of at least 6 months. As will be appreciated, this shelf life may be at frozen temperatures (i.e., −80° C., −25° C., 0° C.), refrigerated (0° C. to 10° C.), or room temperature (20° C. to 32° C.) in liquid or lyophilized form. In further aspects, the highly stabilized formulations of the invention have a shelf life of at least 12, 18, 24, 30, 36, 42, 48, 54, or 60 months.

In further aspects and in accordance with the above, shelf life is determined by a percent activity remaining after storage at any of the above temperatures for any of the above periods of time. In certain embodiments, shelf life means that the formulation retains at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% of furin activity as measured by any of the assays described herein or known in the art as compared to activity prior to storage for any of the above amounts of time at any of the above temperatures.

In one aspect, a highly stabilized formulation of furin (e.g. rfurin) in accordance with the present disclosure includes: (a) 8,000 U/mL-57,000 U/mL furin (e.g., rfurin); (b) 190 mM sodium chloride; (c) 0.92 mM calcium chloride; (d) 10% w/w trehalose dihydrate; (e) 75 ppm polysorbate 80; (f) 45 mM acetic acid; and (g) 46 mM HEPES.

In certain aspects, highly stabilized formulations of the present disclosure include the following components: 47 mM HEPES, 46 mM acetic acid, 195 mM sodium chloride, 0.094 mM calcium chloride, 0.0075% polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0.

In further aspects, highly stabilized formulations of furin, including rfurin, in accordance with the present disclosure include (a) about 5,500 U/mL to 55,000 U/mL; 6,000 U/mL to 50,000 U/mL; 6,500 U/mL to 45,000 U/mL; 7,000 U/mL to 40,000 U/mL; 7,500 U/mL to 35,000 U/mL; 8,000 U/mL to 30,000 U/mL; 8,500 U/mL to 25,000 U/mL; 9,000 U/mL to 20,000 U/mL; 9,500 U/mL to 15,000 U/mL; or 10,000 U/mL furin; (b) about 100 mM to 300 mM, 110 mM to 280 mM, 120 mM to 260 mM, 130 mM to 240 mM, 140 mM to 220 mM, 150 mM to 200 mM, or 160 mM to 180 mM sodium chloride; (c) about 0.5 mM to 9 mM, 1 mM to 8 mM, 1.5 mM to 7 mM, 2 mM to 6 mM, 2.5 mM to 5 mM, or 3 mM to 4.5 mM calcium chloride; (d) about 0.5% to 19%, 1% to 18%, 1.5% to 17%, 2.0% to 16%, 2.5% to 15%, 3.0% to 14%, 3.5% to 13%, 4.0% to 12%, 4.5% to 11%, 5.0% to 10%, 5.5% to 9%, or 6.0% to 8% trehalose dihydrate; (e) about 0.5 ppm to 140 ppm, 1.0 ppm to 130 ppm, 10 ppm to 120 ppm, 20 ppm to 110 ppm, 30 ppm to 100 ppm, 40 ppm to 95 ppm, 50 ppm to 90 ppm, 55 ppm to 85 ppm, 60 ppm to 80 ppm, or 70 ppm to 75 ppm polysorbate 80; (f) about 25 mM to 90 mM, 30 mM to 80 mM, 35 mM to 70 mM, 40 mM to 60 mM, or 45 mM to 50 mM acetic acid, and (g) 15 mM to 95 mM, 20 mM to 90 mM, 25 mM to 85 mM, 30 mM to 80 mM, 35 mM to 75 mM, 40 mM to 70 mM, 45 mM to 65 mM, or 50 mM to 60 mM HEPES.

In one aspect, the present disclosure provides a stabilized aqueous furin composition (e.g., rfurin) comprising: furin (e.g., rfurin), from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 10 mM calcium, a buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar is a pentose or hexose sugar.

In one embodiment, a stabilized aqueous furin composition (e.g., rfurin) comprises: furin (e.g., rfurin), from 100 mM to 300 mM of a pharmaceutically acceptable salt; from 0.5 mM to 2 mM calcium; from 2% to 20% of a sugar or sugar alcohol; from 100 to 200 ppm of a non-ionic surfactant; from 10 to 200 mM buffering agent; and a pH from 5.5 to 7.5. In a specific embodiment, the sugar is a pentose or hexose sugar.

In a specific embodiment, highly stabilized formulations of the invention include the following components: 47 mM HEPES, 46 mM acetic acid, 195 mM sodium chloride, 0.094 mM calcium chloride, 0.0075% polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0.

In another specific embodiment, a highly stabilized formulation of furin (e.g., rfurin) in accordance with the present invention includes: (a) 8,000-57,000 U/mL rfurin; (b) 190 mM sodium chloride; (c) 0.92 mM calcium chloride; (d) 10% w/w trehalose dihydrate; (e) 75 ppm polysorbate 80; (f) 45 mM acetic acid; and (g) 46 mM HEPES.

A. Stabilizing Agents

Advantageously, it was found that the inclusion of sugars, sugar alcohols, and non-ionic surfactants stabilizes aqueous furin (e.g., rfurin) compositions. These effects are demonstrated in the examples provided herein. For example, the addition of these agents increases furin activity retention upon liquid storage, reduces aggregation of furin polypeptides upon liquid storage, reduces the degradation of furin polypeptides upon liquid storage, reduces the loss of furin activity upon agitation of an aqueous furin composition, and reduces aggregation caused by agitation of an aqueous furin composition.

Accordingly, in one embodiment, the present disclosure provides an aqueous furin composition (e.g., a rfurin composition) comprising from 2% to 20% sugar or sugar alcohol and from 10 ppm to 200 ppm non-ionic surfactant. In another embodiment, the composition comprises from 2% to 10% sugar or sugar alcohol and from 10 ppm to 100 ppm non-ionic surfactant. In another embodiment, the composition comprises 10±2% sugar or sugar alcohol and 75±25 ppm non-ionic surfactant. In a specific embodiment, the composition comprises 10% sugar or sugar alcohol and 75 ppm non-ionic surfactant. In yet other embodiments, the composition comprises a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9.

TABLE 1 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 2%-20% 2%-17.5% 2%-15% 2%-12.5% 2%-10% 2%-9% 2%-8% 2%-7% Non-Ionic  10-200 Var. 1 Var. 86 Var. 171 Var. 256 Var. 341 Var. 426 Var. 511 Var. 596 Surfactant  10-175 Var. 2 Var. 87 Var. 172 Var. 257 Var. 342 Var. 427 Var. 512 Var. 597 (ppm)  10-150 Var. 3 Var. 88 Var. 173 Var. 258 Var. 343 Var. 428 Var. 513 Var. 598  10-125 Var. 4 Var. 89 Var. 174 Var. 259 Var. 344 Var. 429 Var. 514 Var. 599  10-100 Var. 5 Var. 90 Var. 175 Var. 260 Var. 345 Var. 430 Var. 515 Var. 600 10-90 Var. 6 Var. 91 Var. 176 Var. 261 Var. 346 Var. 431 Var. 516 Var. 601 10-80 Var. 7 Var. 92 Var. 177 Var. 262 Var. 347 Var. 432 Var. 517 Var. 602 10-75 Var. 8 Var. 93 Var. 178 Var. 263 Var. 348 Var. 433 Var. 518 Var. 603 10-70 Var. 9 Var. 94 Var. 179 Var. 264 Var. 349 Var. 434 Var. 519 Var. 604 10-60 Var. 10 Var. 95 Var. 180 Var. 265 Var. 350 Var. 435 Var. 520 Var. 605 10-50 Var. 11 Var. 96 Var. 181 Var. 266 Var. 351 Var. 436 Var. 521 Var. 606 10-25 Var. 12 Var. 97 Var. 182 Var. 267 Var. 352 Var. 437 Var. 522 Var. 607  25-200 Var. 13 Var. 98 Var. 183 Var. 268 Var. 353 Var. 438 Var. 523 Var. 608  25-175 Var. 14 Var. 99 Var. 184 Var. 269 Var. 354 Var. 439 Var. 524 Var. 609  25-150 Var. 15 Var. 100 Var. 185 Var. 270 Var. 355 Var. 440 Var. 525 Var. 610  25-125 Var. 16 Var. 101 Var. 186 Var. 271 Var. 356 Var. 441 Var. 526 Var. 611  25-100 Var. 17 Var. 102 Var. 187 Var. 272 Var. 357 Var. 442 Var. 527 Var. 612 25-90 Var. 18 Var. 103 Var. 188 Var. 273 Var. 358 Var. 443 Var. 528 Var. 613 25-80 Var. 19 Var. 104 Var. 189 Var. 274 Var. 359 Var. 444 Var. 529 Var. 614 25-70 Var. 20 Var. 105 Var. 190 Var. 275 Var. 360 Var. 445 Var. 530 Var. 615 25-60 Var. 21 Var. 106 Var. 191 Var. 276 Var. 361 Var. 446 Var. 531 Var. 616 25-50 Var. 22 Var. 107 Var. 192 Var. 277 Var. 362 Var. 447 Var. 532 Var. 617  50-200 Var. 23 Var. 108 Var. 193 Var. 278 Var. 363 Var. 448 Var. 533 Var. 618  50-175 Var. 24 Var. 109 Var. 194 Var. 279 Var. 364 Var. 449 Var. 534 Var. 619  50-150 Var. 25 Var. 110 Var. 195 Var. 280 Var. 365 Var. 450 Var. 535 Var. 620  50-125 Var. 26 Var. 111 Var. 196 Var. 281 Var. 366 Var. 451 Var. 536 Var. 621 50-90 Var. 27 Var. 112 Var. 197 Var. 282 Var. 367 Var. 452 Var. 537 Var. 622 50-80 Var. 28 Var. 113 Var. 198 Var. 283 Var. 368 Var. 453 Var. 538 Var. 623  75-200 Var. 29 Var. 114 Var. 199 Var. 284 Var. 369 Var. 454 Var. 539 Var. 624  75-175 Var. 30 Var. 115 Var. 200 Var. 285 Var. 370 Var. 455 Var. 540 Var. 625  75-150 Var. 31 Var. 116 Var. 201 Var. 286 Var. 371 Var. 456 Var. 541 Var. 626 100-200 Var. 32 Var. 117 Var. 202 Var. 287 Var. 372 Var. 457 Var. 542 Var. 627 100-175 Var. 33 Var. 118 Var. 203 Var. 288 Var. 373 Var. 458 Var. 543 Var. 628 50 ± 25 Var. 34 Var. 119 Var. 204 Var. 289 Var. 374 Var. 459 Var. 544 Var. 629 60 ± 25 Var. 35 Var. 120 Var. 205 Var. 290 Var. 375 Var. 460 Var. 545 Var. 630 70 ± 25 Var. 36 Var. 121 Var. 206 Var. 291 Var. 376 Var. 461 Var. 546 Var. 631 75 ± 25 Var. 37 Var. 122 Var. 207 Var. 292 Var. 377 Var. 462 Var. 547 Var. 632 80 ± 25 Var. 38 Var. 123 Var. 208 Var. 293 Var. 378 Var. 463 Var. 548 Var. 633 90 ± 25 Var. 39 Var. 124 Var. 209 Var. 294 Var. 379 Var. 464 Var. 549 Var. 634 100 ± 25  Var. 40 Var. 125 Var. 210 Var. 295 Var. 380 Var. 465 Var. 550 Var. 635 125 ± 25  Var. 41 Var. 126 Var. 211 Var. 296 Var. 381 Var. 466 Var. 551 Var. 636 150 ± 25  Var. 42 Var. 127 Var. 212 Var. 297 Var. 382 Var. 467 Var. 552 Var. 637 175 ± 25  Var. 43 Var. 128 Var. 213 Var. 298 Var. 383 Var. 468 Var. 553 Var. 638 30 ± 10 Var. 44 Var. 129 Var. 214 Var. 299 Var. 384 Var. 469 Var. 554 Var. 639 40 ± 10 Var. 45 Var. 130 Var. 215 Var. 300 Var. 385 Var. 470 Var. 555 Var. 640 50 ± 10 Var. 46 Var. 131 Var. 216 Var. 301 Var. 386 Var. 471 Var. 556 Var. 641 60 ± 10 Var. 47 Var. 132 Var. 217 Var. 302 Var. 387 Var. 472 Var. 557 Var. 642 70 ± 10 Var. 48 Var. 133 Var. 218 Var. 303 Var. 388 Var. 473 Var. 558 Var. 643 75 ± 10 Var. 49 Var. 134 Var. 219 Var. 304 Var. 389 Var. 474 Var. 559 Var. 644 80 ± 10 Var. 50 Var. 135 Var. 220 Var. 305 Var. 390 Var. 475 Var. 560 Var. 645 90 ± 10 Var. 51 Var. 136 Var. 221 Var. 306 Var. 391 Var. 476 Var. 561 Var. 646 100 ± 10  Var. 52 Var. 137 Var. 222 Var. 307 Var. 392 Var. 477 Var. 562 Var. 647 110 ± 10  Var. 53 Var. 138 Var. 223 Var. 308 Var. 393 Var. 478 Var. 563 Var. 648 120 ± 10  Var. 54 Var. 139 Var. 224 Var. 309 Var. 394 Var. 479 Var. 564 Var. 649 125 ± 10  Var. 55 Var. 140 Var. 225 Var. 310 Var. 395 Var. 480 Var. 565 Var. 650 130 ± 10  Var. 56 Var. 141 Var. 226 Var. 311 Var. 396 Var. 481 Var. 566 Var. 651 140 ± 10  Var. 57 Var. 142 Var. 227 Var. 312 Var. 397 Var. 482 Var. 567 Var. 652 150 ± 10  Var. 58 Var. 143 Var. 228 Var. 313 Var. 398 Var. 483 Var. 568 Var. 653 160 ± 10  Var. 59 Var. 144 Var. 229 Var. 314 Var. 399 Var. 484 Var. 569 Var. 654 170 ± 10  Var. 60 Var. 145 Var. 230 Var. 315 Var. 400 Var. 485 Var. 570 Var. 655 175 ± 10  Var. 61 Var. 146 Var. 231 Var. 316 Var. 401 Var. 486 Var. 571 Var. 656 180 ± 10  Var. 62 Var. 147 Var. 232 Var. 317 Var. 402 Var. 487 Var. 572 Var. 657 190 ± 10  Var. 63 Var. 148 Var. 233 Var. 318 Var. 403 Var. 488 Var. 573 Var. 658 25 Var. 64 Var. 149 Var. 234 Var. 319 Var. 404 Var. 489 Var. 574 Var. 659 30 Var. 65 Var. 150 Var. 235 Var. 320 Var. 405 Var. 490 Var. 575 Var. 660 40 Var. 66 Var. 151 Var. 236 Var. 321 Var. 406 Var. 491 Var. 576 Var. 661 50 Var. 67 Var. 152 Var. 237 Var. 322 Var. 407 Var. 492 Var. 577 Var. 662 60 Var. 68 Var. 153 Var. 238 Var. 323 Var. 408 Var. 493 Var. 578 Var. 663 70 Var. 69 Var. 154 Var. 239 Var. 324 Var. 409 Var. 494 Var. 579 Var. 664 75 Var. 70 Var. 155 Var. 240 Var. 325 Var. 410 Var. 495 Var. 580 Var. 665 80 Var. 71 Var. 156 Var. 241 Var. 326 Var. 411 Var. 496 Var. 581 Var. 666 90 Var. 72 Var. 157 Var. 242 Var. 327 Var. 412 Var. 497 Var. 582 Var. 667 100 Var. 73 Var. 158 Var. 243 Var. 328 Var. 413 Var. 498 Var. 583 Var. 668 110 Var. 74 Var. 159 Var. 244 Var. 329 Var. 414 Var. 499 Var. 584 Var. 669 120 Var. 75 Var. 160 Var. 245 Var. 330 Var. 415 Var. 500 Var. 585 Var. 670 125 Var. 76 Var. 161 Var. 246 Var. 331 Var. 416 Var. 501 Var. 586 Var. 671 130 Var. 77 Var. 162 Var. 247 Var. 332 Var. 417 Var. 502 Var. 587 Var. 672 140 Var. 78 Var. 163 Var. 248 Var. 333 Var. 418 Var. 503 Var. 588 Var. 673 150 Var. 79 Var. 164 Var. 249 Var. 334 Var. 419 Var. 504 Var. 589 Var. 674 160 Var. 80 Var. 165 Var. 250 Var. 335 Var. 420 Var. 505 Var. 590 Var. 675 170 Var. 81 Var. 166 Var. 251 Var. 336 Var. 421 Var. 506 Var. 591 Var. 676 175 Var. 82 Var. 167 Var. 252 Var. 337 Var. 422 Var. 507 Var. 592 Var. 677 180 Var. 83 Var. 168 Var. 253 Var. 338 Var. 423 Var. 508 Var. 593 Var. 678 190 Var. 84 Var. 169 Var. 254 Var. 339 Var. 424 Var. 509 Var. 594 Var. 679 200 Var. 85 Var. 170 Var. 255 Var. 340 Var. 425 Var. 510 Var. 595 Var. 680 Var. = Variation

TABLE 2 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 5%-20% 5%-17.5% 5%-15% 5%-12.5% 5%-10% 7.5%-20% 7.5%-7.5% 7.5%-15%

 10-200 Var. 681 Var. 766 Var. 851 Var. 936 Var. 1021 Var. 1106 Var. 1191 Var. 1276 Surfactan  10-175 Var. 682 Var. 767 Var. 852 Var. 937 Var. 1022 Var. 1107 Var. 1192 Var. 1277

 10-150 Var. 683 Var. 768 Var. 853 Var. 938 Var. 1023 Var. 1108 Var. 1193 Var. 1278  10-125 Var. 684 Var. 769 Var. 854 Var. 939 Var. 1024 Var. 1109 Var. 1194 Var. 1279  10-100 Var. 685 Var. 770 Var. 855 Var. 940 Var. 1025 Var. 1110 Var. 1195 Var. 1280 10-90 Var. 686 Var. 771 Var. 856 Var. 941 Var. 1026 Var. 1111 Var. 1196 Var. 1281 10-80 Var. 687 Var. 772 Var. 857 Var. 942 Var. 1027 Var. 1112 Var. 1197 Var. 1282 10-75 Var. 688 Var. 773 Var. 858 Var. 943 Var. 1028 Var. 1113 Var. 1198 Var. 1283 10-70 Var. 689 Var. 774 Var. 859 Var. 944 Var. 1029 Var. 1114 Var. 1199 Var. 1284 10-60 Var. 690 Var. 775 Var. 860 Var. 945 Var. 1030 Var. 1115 Var. 1200 Var. 1285 10-50 Var. 691 Var. 776 Var. 861 Var. 946 Var. 1031 Var. 1116 Var. 1201 Var. 1286 10-25 Var. 692 Var. 777 Var. 862 Var. 947 Var. 1032 Var. 1117 Var. 1202 Var. 1287  25-200 Var. 693 Var. 778 Var. 863 Var. 948 Var. 1033 Var. 1118 Var. 1203 Var. 1288  25-175 Var. 694 Var. 779 Var. 864 Var. 949 Var. 1034 Var. 1119 Var. 1204 Var. 1289  25-150 Var. 695 Var. 780 Var. 865 Var. 950 Var. 1035 Var. 1120 Var. 1205 Var. 1290  25-125 Var. 696 Var. 781 Var. 866 Var. 951 Var. 1036 Var. 1121 Var. 1206 Var. 1291  25-100 Var. 697 Var. 782 Var. 867 Var. 952 Var. 1037 Var. 1122 Var. 1207 Var. 1292 25-90 Var. 698 Var. 783 Var. 868 Var. 953 Var. 1038 Var. 1123 Var. 1208 Var. 1293 25-80 Var. 699 Var. 784 Var. 869 Var. 954 Var. 1039 Var. 1124 Var. 1209 Var. 1294 25-70 Var. 700 Var. 785 Var. 870 Var. 955 Var. 1040 Var. 1125 Var. 1210 Var. 1295 25-60 Var. 701 Var. 786 Var. 871 Var. 956 Var. 1041 Var. 1126 Var. 1211 Var. 1296 25-50 Var. 702 Var. 787 Var. 872 Var. 957 Var. 1042 Var. 1127 Var. 1212 Var. 1297  50-200 Var. 703 Var. 788 Var. 873 Var. 958 Var. 1043 Var. 1128 Var. 1213 Var. 1298  50-175 Var. 704 Var. 789 Var. 874 Var. 959 Var. 1044 Var. 1129 Var. 1214 Var. 1299  50-150 Var. 705 Var. 790 Var. 875 Var. 960 Var. 1045 Var. 1130 Var. 1215 Var. 1300  50-125 Var. 706 Var. 791 Var. 876 Var. 961 Var. 1046 Var. 1131 Var. 1216 Var. 1301 50-90 Var. 707 Var. 792 Var. 877 Var. 962 Var. 1047 Var. 1132 Var. 1217 Var. 1302 50-80 Var. 708 Var. 793 Var. 878 Var. 963 Var. 1048 Var. 1133 Var. 1218 Var. 1303  75-200 Var. 709 Var. 794 Var. 879 Var. 964 Var. 1049 Var. 1134 Var. 1219 Var. 1304  75-175 Var. 710 Var. 795 Var. 880 Var. 965 Var. 1050 Var. 1135 Var. 1220 Var. 1305  75-150 Var. 711 Var. 796 Var. 881 Var. 966 Var. 1051 Var. 1136 Var. 1221 Var. 1306 100-200 Var. 712 Var. 797 Var. 882 Var. 967 Var. 1052 Var. 1137 Var. 1222 Var. 1307 100-175 Var. 713 Var. 798 Var. 883 Var. 968 Var. 1053 Var. 1138 Var. 1223 Var. 1308 50 ± 25 Var. 714 Var. 799 Var. 884 Var. 969 Var. 1054 Var. 1139 Var. 1224 Var. 1309 60 ± 25 Var. 715 Var. 800 Var. 885 Var. 970 Var. 1055 Var. 1140 Var. 1225 Var. 1310 70 ± 25 Var. 716 Var. 801 Var. 886 Var. 971 Var. 1056 Var. 1141 Var. 1226 Var. 1311 75 ± 25 Var. 717 Var. 802 Var. 887 Var. 972 Var. 1057 Var. 1142 Var. 1227 Var. 1312 80 ± 25 Var. 718 Var. 803 Var. 888 Var. 973 Var. 1058 Var. 1143 Var. 1228 Var. 1313 90 ± 25 Var. 719 Var. 804 Var. 889 Var. 974 Var. 1059 Var. 1144 Var. 1229 Var. 1314 100 ± 25  Var. 720 Var. 805 Var. 890 Var. 975 Var. 1060 Var. 1145 Var. 1230 Var. 1315 125 ± 25  Var. 721 Var. 806 Var. 891 Var. 976 Var. 1061 Var. 1146 Var. 1231 Var. 1316 150 ± 25  Var. 722 Var. 807 Var. 892 Var. 977 Var. 1062 Var. 1147 Var. 1232 Var. 1317 175 ± 25  Var. 723 Var. 808 Var. 893 Var. 978 Var. 1063 Var. 1148 Var. 1233 Var. 1318 30 ± 10 Var. 724 Var. 809 Var. 894 Var. 979 Var. 1064 Var. 1149 Var. 1234 Var. 1319 40 ± 10 Var. 725 Var. 810 Var. 895 Var. 980 Var. 1065 Var. 1150 Var. 1235 Var. 1320 50 ± 10 Var. 726 Var. 811 Var. 896 Var. 981 Var. 1066 Var. 1151 Var. 1236 Var. 1321 60 ± 10 Var. 727 Var. 812 Var. 897 Var. 982 Var. 1067 Var. 1152 Var. 1237 Var. 1322 70 ± 10 Var. 728 Var. 813 Var. 898 Var. 983 Var. 1068 Var. 1153 Var. 1238 Var. 1323 75 ± 10 Var. 729 Var. 814 Var. 899 Var. 984 Var. 1069 Var. 1154 Var. 1239 Var. 1324 80 ± 10 Var. 730 Var. 815 Var. 900 Var. 985 Var. 1070 Var. 1155 Var. 1240 Var. 1325 90 ± 10 Var. 731 Var. 816 Var. 901 Var. 986 Var. 1071 Var. 1156 Var. 1241 Var. 1326 100 ± 10  Var. 732 Var. 817 Var. 902 Var. 987 Var. 1072 Var. 1157 Var. 1242 Var. 1327 110 ± 10  Var. 733 Var. 818 Var. 903 Var. 988 Var. 1073 Var. 1158 Var. 1243 Var. 1328 120 ± 10  Var. 734 Var. 819 Var. 904 Var. 989 Var. 1074 Var. 1159 Var. 1244 Var. 1329 125 ± 10  Var. 735 Var. 820 Var. 905 Var. 990 Var. 1075 Var. 1160 Var. 1245 Var. 1330 130 ± 10  Var. 736 Var. 821 Var. 906 Var. 991 Var. 1076 Var. 1161 Var. 1246 Var. 1331 140 ± 10  Var. 737 Var. 822 Var. 907 Var. 992 Var. 1077 Var. 1162 Var. 1247 Var. 1332 150 ± 10  Var. 738 Var. 823 Var. 908 Var. 993 Var. 1078 Var. 1163 Var. 1248 Var. 1333 160 ± 10  Var. 739 Var. 824 Var. 909 Var. 994 Var. 1079 Var. 1164 Var. 1249 Var. 1334 170 ± 10  Var. 740 Var. 825 Var. 910 Var. 995 Var. 1080 Var. 1165 Var. 1250 Var. 1335 175 ± 10  Var. 741 Var. 826 Var. 911 Var. 996 Var. 1081 Var. 1166 Var. 1251 Var. 1336 180 ± 10  Var. 742 Var. 827 Var. 912 Var. 997 Var. 1082 Var. 1167 Var. 1252 Var. 1337 190 ± 10  Var. 743 Var. 828 Var. 913 Var. 998 Var. 1083 Var. 1168 Var. 1253 Var. 1338 25 Var. 744 Var. 829 Var. 914 Var. 999 Var. 1084 Var. 1169 Var. 1254 Var. 1339 30 Var. 745 Var. 830 Var. 915 Var. 1000 Var. 1085 Var. 1170 Var. 1255 Var. 1340 40 Var. 746 Var. 831 Var. 916 Var. 1001 Var. 1086 Var. 1171 Var. 1256 Var. 1341 50 Var. 747 Var. 832 Var. 917 Var. 1002 Var. 1087 Var. 1172 Var. 1257 Var. 1342 60 Var. 748 Var. 833 Var. 918 Var. 1003 Var. 1088 Var. 1173 Var. 1258 Var. 1343 70 Var. 749 Var. 834 Var. 919 Var. 1004 Var. 1089 Var. 1174 Var. 1259 Var. 1344 75 Var. 750 Var. 835 Var. 920 Var. 1005 Var. 1090 Var. 1175 Var. 1260 Var. 1345 80 Var. 751 Var. 836 Var. 921 Var. 1006 Var. 1091 Var. 1176 Var. 1261 Var. 1346 90 Var. 752 Var. 837 Var. 922 Var. 1007 Var. 1092 Var. 1177 Var. 1262 Var. 1347 100 Var. 753 Var. 838 Var. 923 Var. 1008 Var. 1093 Var. 1178 Var. 1263 Var. 1348 110 Var. 754 Var. 839 Var. 924 Var. 1009 Var. 1094 Var. 1179 Var. 1264 Var. 1349 120 Var. 755 Var. 840 Var. 925 Var. 1010 Var. 1095 Var. 1180 Var. 1265 Var. 1350 125 Var. 756 Var. 841 Var. 926 Var. 1011 Var. 1096 Var. 1181 Var. 1266 Var. 1351 130 Var. 757 Var. 842 Var. 927 Var. 1012 Var. 1097 Var. 1182 Var. 1267 Var. 1352 140 Var. 758 Var. 843 Var. 928 Var. 1013 Var. 1098 Var. 1183 Var. 1268 Var. 1353 150 Var. 759 Var. 844 Var. 929 Var. 1014 Var. 1099 Var. 1184 Var. 1269 Var. 1354 160 Var. 760 Var. 845 Var. 930 Var. 1015 Var. 1100 Var. 1185 Var. 1270 Var. 1355 170 Var. 761 Var. 846 Var. 931 Var. 1016 Var. 1101 Var. 1186 Var. 1271 Var. 1356 175 Var. 762 Var. 847 Var. 932 Var. 1017 Var. 1102 Var. 1187 Var. 1272 Var. 1357 180 Var. 763 Var. 848 Var. 933 Var. 1018 Var. 1103 Var. 1188 Var. 1273 Var. 1358 190 Var. 764 Var. 849 Var. 934 Var. 1019 Var. 1104 Var. 1189 Var. 1274 Var. 1359 200 Var. 765 Var. 850 Var. 935 Var. 1020 Var. 1105 Var. 1190 Var. 1275 Var. 1360 Var. = Variation

indicates data missing or illegible when filed

TABLE 3 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 7.5%-12.5% 10%-20% 10%-17.5% 10%-15% 4 ± 2% 5 ± 2% 6 ± 2% 7 ± 2% Non-Ionic  10-200 Var. 1361 Var. 1446 Var. 1531 Var. 1616 Var. 1701 Var. 1786 Var. 1871 Var. 1956 Surfactant  10-175 Var. 1362 Var. 1447 Var. 1532 Var. 1617 Var. 1702 Var. 1787 Var. 1872 Var. 1957 (ppm)  10-150 Var. 1363 Var. 1448 Var. 1533 Var. 1618 Var. 1703 Var. 1788 Var. 1873 Var. 1958  10-125 Var. 1364 Var. 1449 Var. 1534 Var. 1619 Var. 1704 Var. 1789 Var. 1874 Var. 1959  10-100 Var. 1365 Var. 1450 Var. 1535 Var. 1620 Var. 1705 Var. 1790 Var. 1875 Var. 1960 10-90 Var. 1366 Var. 1451 Var. 1536 Var. 1621 Var. 1706 Var. 1791 Var. 1876 Var. 1961 10-80 Var. 1367 Var. 1452 Var. 1537 Var. 1622 Var. 1707 Var. 1792 Var. 1877 Var. 1962 10-75 Var. 1368 Var. 1453 Var. 1538 Var. 1623 Var. 1708 Var. 1793 Var. 1878 Var. 1963 10-70 Var. 1369 Var. 1454 Var. 1539 Var. 1624 Var. 1709 Var. 1794 Var. 1879 Var. 1964 10-60 Var. 1370 Var. 1455 Var. 1540 Var. 1625 Var. 1710 Var. 1795 Var. 1880 Var. 1965 10-50 Var. 1371 Var. 1456 Var. 1541 Var. 1626 Var. 1711 Var. 1796 Var. 1881 Var. 1966 10-25 Var. 1372 Var. 1457 Var. 1542 Var. 1627 Var. 1712 Var. 1797 Var. 1882 Var. 1967  25-200 Var. 1373 Var. 1458 Var. 1543 Var. 1628 Var. 1713 Var. 1798 Var. 1883 Var. 1968  25-175 Var. 1374 Var. 1459 Var. 1544 Var. 1629 Var. 1714 Var. 1799 Var. 1884 Var. 1969  25-150 Var. 1375 Var. 1460 Var. 1545 Var. 1630 Var. 1715 Var. 1800 Var. 1885 Var. 1970  25-125 Var. 1376 Var. 1461 Var. 1546 Var. 1631 Var. 1716 Var. 1801 Var. 1886 Var. 1971  25-100 Var. 1377 Var. 1462 Var. 1547 Var. 1632 Var. 1717 Var. 1802 Var. 1887 Var. 1972 25-90 Var. 1378 Var. 1463 Var. 1548 Var. 1633 Var. 1718 Var. 1803 Var. 1888 Var. 1973 25-80 Var. 1379 Var. 1464 Var. 1549 Var. 1634 Var. 1719 Var. 1804 Var. 1889 Var. 1974 25-70 Var. 1380 Var. 1465 Var. 1550 Var. 1635 Var. 1720 Var. 1805 Var. 1890 Var. 1975 25-60 Var. 1381 Var. 1466 Var. 1551 Var. 1636 Var. 1721 Var. 1806 Var. 1891 Var. 1976 25-50 Var. 1382 Var. 1467 Var. 1552 Var. 1637 Var. 1722 Var. 1807 Var. 1892 Var. 1977  50-200 Var. 1383 Var. 1468 Var. 1553 Var. 1638 Var. 1723 Var. 1808 Var. 1893 Var. 1978  50-175 Var. 1384 Var. 1469 Var. 1554 Var. 1639 Var. 1724 Var. 1809 Var. 1894 Var. 1979  50-150 Var. 1385 Var. 1470 Var. 1555 Var. 1640 Var. 1725 Var. 1810 Var. 1895 Var. 1980  50-125 Var. 1386 Var. 1471 Var. 1556 Var. 1641 Var. 1726 Var. 1811 Var. 1896 Var. 1981 50-90 Var. 1387 Var. 1472 Var. 1557 Var. 1642 Var. 1727 Var. 1812 Var. 1897 Var. 1982 50-80 Var. 1388 Var. 1473 Var. 1558 Var. 1643 Var. 1728 Var. 1813 Var. 1898 Var. 1983  75-200 Var. 1389 Var. 1474 Var. 1559 Var. 1644 Var. 1729 Var. 1814 Var. 1899 Var. 1984  75-175 Var. 1390 Var. 1475 Var. 1560 Var. 1645 Var. 1730 Var. 1815 Var. 1900 Var. 1985  75-150 Var. 1391 Var. 1476 Var. 1561 Var. 1646 Var. 1731 Var. 1816 Var. 1901 Var. 1986 100-200 Var. 1392 Var. 1477 Var. 1562 Var. 1647 Var. 1732 Var. 1817 Var. 1902 Var. 1987 100-175 Var. 1393 Var. 1478 Var. 1563 Var. 1648 Var. 1733 Var. 1818 Var. 1903 Var. 1988 50 ± 25 Var. 1394 Var. 1479 Var. 1564 Var. 1649 Var. 1734 Var. 1819 Var. 1904 Var. 1989 60 ± 25 Var. 1395 Var. 1480 Var. 1565 Var. 1650 Var. 1735 Var. 1820 Var. 1905 Var. 1990 70 ± 25 Var. 1396 Var. 1481 Var. 1566 Var. 1651 Var. 1736 Var. 1821 Var. 1906 Var. 1991 75 ± 25 Var. 1397 Var. 1482 Var. 1567 Var. 1652 Var. 1737 Var. 1822 Var. 1907 Var. 1992 80 ± 25 Var. 1398 Var. 1483 Var. 1568 Var. 1653 Var. 1738 Var. 1823 Var. 1908 Var. 1993 90 ± 25 Var. 1399 Var. 1484 Var. 1569 Var. 1654 Var. 1739 Var. 1824 Var. 1909 Var. 1994 100 ± 25  Var. 1400 Var. 1485 Var. 1570 Var. 1655 Var. 1740 Var. 1825 Var. 1910 Var. 1995 125 ± 25  Var. 1401 Var. 1486 Var. 1571 Var. 1656 Var. 1741 Var. 1826 Var. 1911 Var. 1996 150 ± 25  Var. 1402 Var. 1487 Var. 1572 Var. 1657 Var. 1742 Var. 1827 Var. 1912 Var. 1997 175 ± 25  Var. 1403 Var. 1488 Var. 1573 Var. 1658 Var. 1743 Var. 1828 Var. 1913 Var. 1998 30 ± 10 Var. 1404 Var. 1489 Var. 1574 Var. 1659 Var. 1744 Var. 1829 Var. 1914 Var. 1999 40 ± 10 Var. 1405 Var. 1490 Var. 1575 Var. 1660 Var. 1745 Var. 1830 Var. 1915 Var. 2000 50 ± 10 Var. 1406 Var. 1491 Var. 1576 Var. 1661 Var. 1746 Var. 1831 Var. 1916 Var. 2001 60 ± 10 Var. 1407 Var. 1492 Var. 1577 Var. 1662 Var. 1747 Var. 1832 Var. 1917 Var. 2002 70 ± 10 Var. 1408 Var. 1493 Var. 1578 Var. 1663 Var. 1748 Var. 1833 Var. 1918 Var. 2003 75 ± 10 Var. 1409 Var. 1494 Var. 1579 Var. 1664 Var. 1749 Var. 1834 Var. 1919 Var. 2004 80 ± 10 Var. 1410 Var. 1495 Var. 1580 Var. 1665 Var. 1750 Var. 1835 Var. 1920 Var. 2005 90 ± 10 Var. 1411 Var. 1496 Var. 1581 Var. 1666 Var. 1751 Var. 1836 Var. 1921 Var. 2006 100 ± 10  Var. 1412 Var. 1497 Var. 1582 Var. 1667 Var. 1752 Var. 1837 Var. 1922 Var. 2007 110 ± 10  Var. 1413 Var. 1498 Var. 1583 Var. 1668 Var. 1753 Var. 1838 Var. 1923 Var. 2008 120 ± 10  Var. 1414 Var. 1499 Var. 1584 Var. 1669 Var. 1754 Var. 1839 Var. 1924 Var. 2009 125 ± 10  Var. 1415 Var. 1500 Var. 1585 Var. 1670 Var. 1755 Var. 1840 Var. 1925 Var. 2010 130 ± 10  Var. 1416 Var. 1501 Var. 1586 Var. 1671 Var. 1756 Var. 1841 Var. 1926 Var. 2011 140 ± 10  Var. 1417 Var. 1502 Var. 1587 Var. 1672 Var. 1757 Var. 1842 Var. 1927 Var. 2012 150 ± 10  Var. 1418 Var. 1503 Var. 1588 Var. 1673 Var. 1758 Var. 1843 Var. 1928 Var. 2013 160 ± 10  Var. 1419 Var. 1504 Var. 1589 Var. 1674 Var. 1759 Var. 1844 Var. 1929 Var. 2014 170 ± 10  Var. 1420 Var. 1505 Var. 1590 Var. 1675 Var. 1760 Var. 1845 Var. 1930 Var. 2015 175 ± 10  Var. 1421 Var. 1506 Var. 1591 Var. 1676 Var. 1761 Var. 1846 Var. 1931 Var. 2016 180 ± 10  Var. 1422 Var. 1507 Var. 1592 Var. 1677 Var. 1762 Var. 1847 Var. 1932 Var. 2017 190 ± 10  Var. 1423 Var. 1508 Var. 1593 Var. 1678 Var. 1763 Var. 1848 Var. 1933 Var. 2018 25 Var. 1424 Var. 1509 Var. 1594 Var. 1679 Var. 1764 Var. 1849 Var. 1934 Var. 2019 30 Var. 1425 Var. 1510 Var. 1595 Var. 1680 Var. 1765 Var. 1850 Var. 1935 Var. 2020 40 Var. 1426 Var. 1511 Var. 1596 Var. 1681 Var. 1766 Var. 1851 Var. 1936 Var. 2021 50 Var. 1427 Var. 1512 Var. 1597 Var. 1682 Var. 1767 Var. 1852 Var. 1937 Var. 2022 60 Var. 1428 Var. 1513 Var. 1598 Var. 1683 Var. 1768 Var. 1853 Var. 1938 Var. 2023 70 Var. 1429 Var. 1514 Var. 1599 Var. 1684 Var. 1769 Var. 1854 Var. 1939 Var. 2024 75 Var. 1430 Var. 1515 Var. 1600 Var. 1685 Var. 1770 Var. 1855 Var. 1940 Var. 2025 80 Var. 1431 Var. 1516 Var. 1601 Var. 1686 Var. 1771 Var. 1856 Var. 1941 Var. 2026 90 Var. 1432 Var. 1517 Var. 1602 Var. 1687 Var. 1772 Var. 1857 Var. 1942 Var. 2027 100 Var. 1433 Var. 1518 Var. 1603 Var. 1688 Var. 1773 Var. 1858 Var. 1943 Var. 2028 110 Var. 1434 Var. 1519 Var. 1604 Var. 1689 Var. 1774 Var. 1859 Var. 1944 Var. 2029 120 Var. 1435 Var. 1520 Var. 1605 Var. 1690 Var. 1775 Var. 1860 Var. 1945 Var. 2030 125 Var. 1436 Var. 1521 Var. 1606 Var. 1691 Var. 1776 Var. 1861 Var. 1946 Var. 2031 130 Var. 1437 Var. 1522 Var. 1607 Var. 1692 Var. 1777 Var. 1862 Var. 1947 Var. 2032 140 Var. 1438 Var. 1523 Var. 1608 Var. 1693 Var. 1778 Var. 1863 Var. 1948 Var. 2033 150 Var. 1439 Var. 1524 Var. 1609 Var. 1694 Var. 1779 Var. 1864 Var. 1949 Var. 2034 160 Var. 1440 Var. 1525 Var. 1610 Var. 1695 Var. 1780 Var. 1865 Var. 1950 Var. 2035 170 Var. 1441 Var. 1526 Var. 1611 Var. 1696 Var. 1781 Var. 1866 Var. 1951 Var. 2036 175 Var. 1442 Var. 1527 Var. 1612 Var. 1697 Var. 1782 Var. 1867 Var. 1952 Var. 2037 180 Var. 1443 Var. 1528 Var. 1613 Var. 1698 Var. 1783 Var. 1868 Var. 1953 Var. 2038 190 Var. 1444 Var. 1529 Var. 1614 Var. 1699 Var. 1784 Var. 1869 Var. 1954 Var. 2039 200 Var. 1445 Var. 1530 Var. 1615 Var. 1700 Var. 1785 Var. 1870 Var. 1955 Var. 2040 Var. = Variation

TABLE 4 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 8 ± 2% 9 ± 2% 10 ± 2% 11 ± 2% 12 ± 2% 13 ± 2% 14 ± 2% 15 ± 2% Non-Ionic  10-200 Var. 2041 Var. 2126 Var. 2211 Var. 2296 Var. 2381 Var. 2466 Var. 2551 Var. 2636 Surfactant  10-175 Var. 2042 Var. 2127 Var. 2212 Var. 2297 Var. 2382 Var. 2467 Var. 2552 Var. 2637 (ppm)  10-150 Var. 2043 Var. 2128 Var. 2213 Var. 2298 Var. 2383 Var. 2468 Var. 2553 Var. 2638  10-125 Var. 2044 Var. 2129 Var. 2214 Var. 2299 Var. 2384 Var. 2469 Var. 2554 Var. 2639  10-100 Var. 2045 Var. 2130 Var. 2215 Var. 2300 Var. 2385 Var. 2470 Var. 2555 Var. 2640 10-90 Var. 2046 Var. 2131 Var. 2216 Var. 2301 Var. 2386 Var. 2471 Var. 2556 Var. 2641 10-80 Var. 2047 Var. 2132 Var. 2217 Var. 2302 Var. 2387 Var. 2472 Var. 2557 Var. 2642 10-75 Var. 2048 Var. 2133 Var. 2218 Var. 2303 Var. 2388 Var. 2473 Var. 2558 Var. 2643 10-70 Var. 2049 Var. 2134 Var. 2219 Var. 2304 Var. 2389 Var. 2474 Var. 2559 Var. 2644 10-60 Var. 2050 Var. 2135 Var. 2220 Var. 2305 Var. 2390 Var. 2475 Var. 2560 Var. 2645 10-50 Var. 2051 Var. 2136 Var. 2221 Var. 2306 Var. 2391 Var. 2476 Var. 2561 Var. 2646 10-25 Var. 2052 Var. 2137 Var. 2222 Var. 2307 Var. 2392 Var. 2477 Var. 2562 Var. 2647  25-200 Var. 2053 Var. 2138 Var. 2223 Var. 2308 Var. 2393 Var. 2478 Var. 2563 Var. 2648  25-175 Var. 2054 Var. 2139 Var. 2224 Var. 2309 Var. 2394 Var. 2479 Var. 2564 Var. 2649  25-150 Var. 2055 Var. 2140 Var. 2225 Var. 2310 Var. 2395 Var. 2480 Var. 2565 Var. 2650  25-125 Var. 2056 Var. 2141 Var. 2226 Var. 2311 Var. 2396 Var. 2481 Var. 2566 Var. 2651  25-100 Var. 2057 Var. 2142 Var. 2227 Var. 2312 Var. 2397 Var. 2482 Var. 2567 Var. 2652 25-90 Var. 2058 Var. 2143 Var. 2228 Var. 2313 Var. 2398 Var. 2483 Var. 2568 Var. 2653 25-80 Var. 2059 Var. 2144 Var. 2229 Var. 2314 Var. 2399 Var. 2484 Var. 2569 Var. 2654 25-70 Var. 2060 Var. 2145 Var. 2230 Var. 2315 Var. 2400 Var. 2485 Var. 2570 Var. 2655 25-60 Var. 2061 Var. 2146 Var. 2231 Var. 2316 Var. 2401 Var. 2486 Var. 2571 Var. 2656 25-50 Var. 2062 Var. 2147 Var. 2232 Var. 2317 Var. 2402 Var. 2487 Var. 2572 Var. 2657  50-200 Var. 2063 Var. 2148 Var. 2233 Var. 2318 Var. 2403 Var. 2488 Var. 2573 Var. 2658  50-175 Var. 2064 Var. 2149 Var. 2234 Var. 2319 Var. 2404 Var. 2489 Var. 2574 Var. 2659  50-150 Var. 2065 Var. 2150 Var. 2235 Var. 2320 Var. 2405 Var. 2490 Var. 2575 Var. 2660  50-125 Var. 2066 Var. 2151 Var. 2236 Var. 2321 Var. 2406 Var. 2491 Var. 2576 Var. 2661 50-90 Var. 2067 Var. 2152 Var. 2237 Var. 2322 Var. 2407 Var. 2492 Var. 2577 Var. 2662 50-80 Var. 2068 Var. 2153 Var. 2238 Var. 2323 Var. 2408 Var. 2493 Var. 2578 Var. 2663  75-200 Var. 2069 Var. 2154 Var. 2239 Var. 2324 Var. 2409 Var. 2494 Var. 2579 Var. 2664  75-175 Var. 2070 Var. 2155 Var. 2240 Var. 2325 Var. 2410 Var. 2495 Var. 2580 Var. 2665  75-150 Var. 2071 Var. 2156 Var. 2241 Var. 2326 Var. 2411 Var. 2496 Var. 2581 Var. 2666 100-200 Var. 2072 Var. 2157 Var. 2242 Var. 2327 Var. 2412 Var. 2497 Var. 2582 Var. 2667 100-175 Var. 2073 Var. 2158 Var. 2243 Var. 2328 Var. 2413 Var. 2498 Var. 2583 Var. 2668 50 ± 25 Var. 2074 Var. 2159 Var. 2244 Var. 2329 Var. 2414 Var. 2499 Var. 2584 Var. 2669 60 ± 25 Var. 2075 Var. 2160 Var. 2245 Var. 2330 Var. 2415 Var. 2500 Var. 2585 Var. 2670 70 ± 25 Var. 2076 Var. 2161 Var. 2246 Var. 2331 Var. 2416 Var. 2501 Var. 2586 Var. 2671 75 ± 25 Var. 2077 Var. 2162 Var. 2247 Var. 2332 Var. 2417 Var. 2502 Var. 2587 Var. 2672 80 ± 25 Var. 2078 Var. 2163 Var. 2248 Var. 2333 Var. 2418 Var. 2503 Var. 2588 Var. 2673 90 ± 25 Var. 2079 Var. 2164 Var. 2249 Var. 2334 Var. 2419 Var. 2504 Var. 2589 Var. 2674 100 ± 25  Var. 2080 Var. 2165 Var. 2250 Var. 2335 Var. 2420 Var. 2505 Var. 2590 Var. 2675 125 ± 25  Var. 2081 Var. 2166 Var. 2251 Var. 2336 Var. 2421 Var. 2506 Var. 2591 Var. 2676 150 ± 25  Var. 2082 Var. 2167 Var. 2252 Var. 2337 Var. 2422 Var. 2507 Var. 2592 Var. 2677 175 ± 25  Var. 2083 Var. 2168 Var. 2253 Var. 2338 Var. 2423 Var. 2508 Var. 2593 Var. 2678 30 ± 10 Var. 2084 Var. 2169 Var. 2254 Var. 2339 Var. 2424 Var. 2509 Var. 2594 Var. 2679 40 ± 10 Var. 2085 Var. 2170 Var. 2255 Var. 2340 Var. 2425 Var. 2510 Var. 2595 Var. 2680 50 ± 10 Var. 2086 Var. 2171 Var. 2256 Var. 2341 Var. 2426 Var. 2511 Var. 2596 Var. 2681 60 ± 10 Var. 2087 Var. 2172 Var. 2257 Var. 2342 Var. 2427 Var. 2512 Var. 2597 Var. 2682 70 ± 10 Var. 2088 Var. 2173 Var. 2258 Var. 2343 Var. 2428 Var. 2513 Var. 2598 Var. 2683 75 ± 10 Var. 2089 Var. 2174 Var. 2259 Var. 2344 Var. 2429 Var. 2514 Var. 2599 Var. 2684 80 ± 10 Var. 2090 Var. 2175 Var. 2260 Var. 2345 Var. 2430 Var. 2515 Var. 2600 Var. 2685 90 ± 10 Var. 2091 Var. 2176 Var. 2261 Var. 2346 Var. 2431 Var. 2516 Var. 2601 Var. 2686 100 ± 10  Var. 2092 Var. 2177 Var. 2262 Var. 2347 Var. 2432 Var. 2517 Var. 2602 Var. 2687 110 ± 10  Var. 2093 Var. 2178 Var. 2263 Var. 2348 Var. 2433 Var. 2518 Var. 2603 Var. 2688 120 ± 10  Var. 2094 Var. 2179 Var. 2264 Var. 2349 Var. 2434 Var. 2519 Var. 2604 Var. 2689 125 ± 10  Var. 2095 Var. 2180 Var. 2265 Var. 2350 Var. 2435 Var. 2520 Var. 2605 Var. 2690 130 ± 10  Var. 2096 Var. 2181 Var. 2266 Var. 2351 Var. 2436 Var. 2521 Var. 2606 Var. 2691 140 ± 10  Var. 2097 Var. 2182 Var. 2267 Var. 2352 Var. 2437 Var. 2522 Var. 2607 Var. 2692 150 ± 10  Var. 2098 Var. 2183 Var. 2268 Var. 2353 Var. 2438 Var. 2523 Var. 2608 Var. 2693 160 ± 10  Var. 2099 Var. 2184 Var. 2269 Var. 2354 Var. 2439 Var. 2524 Var. 2609 Var. 2694 170 ± 10  Var. 2100 Var. 2185 Var. 2270 Var. 2355 Var. 2440 Var. 2525 Var. 2610 Var. 2695 175 ± 10  Var. 2101 Var. 2186 Var. 2271 Var. 2356 Var. 2441 Var. 2526 Var. 2611 Var. 2696 180 ± 10  Var. 2102 Var. 2187 Var. 2272 Var. 2357 Var. 2442 Var. 2527 Var. 2612 Var. 2697 190 ± 10  Var. 2103 Var. 2188 Var. 2273 Var. 2358 Var. 2443 Var. 2528 Var. 2613 Var. 2698 25 Var. 2104 Var. 2189 Var. 2274 Var. 2359 Var. 2444 Var. 2529 Var. 2614 Var. 2699 30 Var. 2105 Var. 2190 Var. 2275 Var. 2360 Var. 2445 Var. 2530 Var. 2615 Var. 2700 40 Var. 2106 Var. 2191 Var. 2276 Var. 2361 Var. 2446 Var. 2531 Var. 2616 Var. 2701 50 Var. 2107 Var. 2192 Var. 2277 Var. 2362 Var. 2447 Var. 2532 Var. 2617 Var. 2702 60 Var. 2108 Var. 2193 Var. 2278 Var. 2363 Var. 2448 Var. 2533 Var. 2618 Var. 2703 70 Var. 2109 Var. 2194 Var. 2279 Var. 2364 Var. 2449 Var. 2534 Var. 2619 Var. 2704 75 Var. 2110 Var. 2195 Var. 2280 Var. 2365 Var. 2450 Var. 2535 Var. 2620 Var. 2705 80 Var. 2111 Var. 2196 Var. 2281 Var. 2366 Var. 2451 Var. 2536 Var. 2621 Var. 2706 90 Var. 2112 Var. 2197 Var. 2282 Var. 2367 Var. 2452 Var. 2537 Var. 2622 Var. 2707 100 Var. 2113 Var. 2198 Var. 2283 Var. 2368 Var. 2453 Var. 2538 Var. 2623 Var. 2708 110 Var. 2114 Var. 2199 Var. 2284 Var. 2369 Var. 2454 Var. 2539 Var. 2624 Var. 2709 120 Var. 2115 Var. 2200 Var. 2285 Var. 2370 Var. 2455 Var. 2540 Var. 2625 Var. 2710 125 Var. 2116 Var. 2201 Var. 2286 Var. 2371 Var. 2456 Var. 2541 Var. 2626 Var. 2711 130 Var. 2117 Var. 2202 Var. 2287 Var. 2372 Var. 2457 Var. 2542 Var. 2627 Var. 2712 140 Var. 2118 Var. 2203 Var. 2288 Var. 2373 Var. 2458 Var. 2543 Var. 2628 Var. 2713 150 Var. 2119 Var. 2204 Var. 2289 Var. 2374 Var. 2459 Var. 2544 Var. 2629 Var. 2714 160 Var. 2120 Var. 2205 Var. 2290 Var. 2375 Var. 2460 Var. 2545 Var. 2630 Var. 2715 170 Var. 2121 Var. 2206 Var. 2291 Var. 2376 Var. 2461 Var. 2546 Var. 2631 Var. 2716 175 Var. 2122 Var. 2207 Var. 2292 Var. 2377 Var. 2462 Var. 2547 Var. 2632 Var. 2717 180 Var. 2123 Var. 2208 Var. 2293 Var. 2378 Var. 2463 Var. 2548 Var. 2633 Var. 2718 190 Var. 2124 Var. 2209 Var. 2294 Var. 2379 Var. 2464 Var. 2549 Var. 2634 Var. 2719 200 Var. 2125 Var. 2210 Var. 2295 Var. 2380 Var. 2465 Var. 2550 Var. 2635 Var. 2720 Var. = Variation

TABLE 5 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 16 ± 2% 17 ± 2% 18 ± 2% 3 ± 1% 4 ± 1% 5 ± 1% 6 ± 1% 7 ± 1% Non-Ionic  10-200 Var. 2721 Var. 2806 Var. 2891 Var. 2976 Var. 3061 Var. 3146 Var. 3231 Var. 3316 Surfactant  10-175 Var. 2722 Var. 2807 Var. 2892 Var. 2977 Var. 3062 Var. 3147 Var. 3232 Var. 3317 (ppm)  10-150 Var. 2723 Var. 2808 Var. 2893 Var. 2978 Var. 3063 Var. 3148 Var. 3233 Var. 3318  10-125 Var. 2724 Var. 2809 Var. 2894 Var. 2979 Var. 3064 Var. 3149 Var. 3234 Var. 3319  10-100 Var. 2725 Var. 2810 Var. 2895 Var. 2980 Var. 3065 Var. 3150 Var. 3235 Var. 3320 10-90 Var. 2726 Var. 2811 Var. 2896 Var. 2981 Var. 3066 Var. 3151 Var. 3236 Var. 3321 10-80 Var. 2727 Var. 2812 Var. 2897 Var. 2982 Var. 3067 Var. 3152 Var. 3237 Var. 3322 10-75 Var. 2728 Var. 2813 Var. 2898 Var. 2983 Var. 3068 Var. 3153 Var. 3238 Var. 3323 10-70 Var. 2729 Var. 2814 Var. 2899 Var. 2984 Var. 3069 Var. 3154 Var. 3239 Var. 3324 10-60 Var. 2730 Var. 2815 Var. 2900 Var. 2985 Var. 3070 Var. 3155 Var. 3240 Var. 3325 10-50 Var. 2731 Var. 2816 Var. 2901 Var. 2986 Var. 3071 Var. 3156 Var. 3241 Var. 3326 10-25 Var. 2732 Var. 2817 Var. 2902 Var. 2987 Var. 3072 Var. 3157 Var. 3242 Var. 3327  25-200 Var. 2733 Var. 2818 Var. 2903 Var. 2988 Var. 3073 Var. 3158 Var. 3243 Var. 3328  25-175 Var. 2734 Var. 2819 Var. 2904 Var. 2989 Var. 3074 Var. 3159 Var. 3244 Var. 3329  25-150 Var. 2735 Var. 2820 Var. 2905 Var. 2990 Var. 3075 Var. 3160 Var. 3245 Var. 3330  25-125 Var. 2736 Var. 2821 Var. 2906 Var. 2991 Var. 3076 Var. 3161 Var. 3246 Var. 3331  25-100 Var. 2737 Var. 2822 Var. 2907 Var. 2992 Var. 3077 Var. 3162 Var. 3247 Var. 3332 25-90 Var. 2738 Var. 2823 Var. 2908 Var. 2993 Var. 3078 Var. 3163 Var. 3248 Var. 3333 25-80 Var. 2739 Var. 2824 Var. 2909 Var. 2994 Var. 3079 Var. 3164 Var. 3249 Var. 3334 25-70 Var. 2740 Var. 2825 Var. 2910 Var. 2995 Var. 3080 Var. 3165 Var. 3250 Var. 3335 25-60 Var. 2741 Var. 2826 Var. 2911 Var. 2996 Var. 3081 Var. 3166 Var. 3251 Var. 3336 25-50 Var. 2742 Var. 2827 Var. 2912 Var. 2997 Var. 3082 Var. 3167 Var. 3252 Var. 3337  50-200 Var. 2743 Var. 2828 Var. 2913 Var. 2998 Var. 3083 Var. 3168 Var. 3253 Var. 3338  50-175 Var. 2744 Var. 2829 Var. 2914 Var. 2999 Var. 3084 Var. 3169 Var. 3254 Var. 3339  50-150 Var. 2745 Var. 2830 Var. 2915 Var. 3000 Var. 3085 Var. 3170 Var. 3255 Var. 3340  50-125 Var. 2746 Var. 2831 Var. 2916 Var. 3001 Var. 3086 Var. 3171 Var. 3256 Var. 3341 50-90 Var. 2747 Var. 2832 Var. 2917 Var. 3002 Var. 3087 Var. 3172 Var. 3257 Var. 3342 50-80 Var. 2748 Var. 2833 Var. 2918 Var. 3003 Var. 3088 Var. 3173 Var. 3258 Var. 3343  75-200 Var. 2749 Var. 2834 Var. 2919 Var. 3004 Var. 3089 Var. 3174 Var. 3259 Var. 3344  75-175 Var. 2750 Var. 2835 Var. 2920 Var. 3005 Var. 3090 Var. 3175 Var. 3260 Var. 3345  75-150 Var. 2751 Var. 2836 Var. 2921 Var. 3006 Var. 3091 Var. 3176 Var. 3261 Var. 3346 100-200 Var. 2752 Var. 2837 Var. 2922 Var. 3007 Var. 3092 Var. 3177 Var. 3262 Var. 3347 100-175 Var. 2753 Var. 2838 Var. 2923 Var. 3008 Var. 3093 Var. 3178 Var. 3263 Var. 3348 50 ± 25 Var. 2754 Var. 2839 Var. 2924 Var. 3009 Var. 3094 Var. 3179 Var. 3264 Var. 3349 60 ± 25 Var. 2755 Var. 2840 Var. 2925 Var. 3010 Var. 3095 Var. 3180 Var. 3265 Var. 3350 70 ± 25 Var. 2756 Var. 2841 Var. 2926 Var. 3011 Var. 3096 Var. 3181 Var. 3266 Var. 3351 75 ± 25 Var. 2757 Var. 2842 Var. 2927 Var. 3012 Var. 3097 Var. 3182 Var. 3267 Var. 3352 80 ± 25 Var. 2758 Var. 2843 Var. 2928 Var. 3013 Var. 3098 Var. 3183 Var. 3268 Var. 3353 90 ± 25 Var. 2759 Var. 2844 Var. 2929 Var. 3014 Var. 3099 Var. 3184 Var. 3269 Var. 3354 100 ± 25  Var. 2760 Var. 2845 Var. 2930 Var. 3015 Var. 3100 Var. 3185 Var. 3270 Var. 3355 125 ± 25  Var. 2761 Var. 2846 Var. 2931 Var. 3016 Var. 3101 Var. 3186 Var. 3271 Var. 3356 150 ± 25  Var. 2762 Var. 2847 Var. 2932 Var. 3017 Var. 3102 Var. 3187 Var. 3272 Var. 3357 175 ± 25  Var. 2763 Var. 2848 Var. 2933 Var. 3018 Var. 3103 Var. 3188 Var. 3273 Var. 3358 30 ± 10 Var. 2764 Var. 2849 Var. 2934 Var. 3019 Var. 3104 Var. 3189 Var. 3274 Var. 3359 40 ± 10 Var. 2765 Var. 2850 Var. 2935 Var. 3020 Var. 3105 Var. 3190 Var. 3275 Var. 3360 50 ± 10 Var. 2766 Var. 2851 Var. 2936 Var. 3021 Var. 3106 Var. 3191 Var. 3276 Var. 3361 60 ± 10 Var. 2767 Var. 2852 Var. 2937 Var. 3022 Var. 3107 Var. 3192 Var. 3277 Var. 3362 70 ± 10 Var. 2768 Var. 2853 Var. 2938 Var. 3023 Var. 3108 Var. 3193 Var. 3278 Var. 3363 75 ± 10 Var. 2769 Var. 2854 Var. 2939 Var. 3024 Var. 3109 Var. 3194 Var. 3279 Var. 3364 80 ± 10 Var. 2770 Var. 2855 Var. 2940 Var. 3025 Var. 3110 Var. 3195 Var. 3280 Var. 3365 90 ± 10 Var. 2771 Var. 2856 Var. 2941 Var. 3026 Var. 3111 Var. 3196 Var. 3281 Var. 3366 100 ± 10  Var. 2772 Var. 2857 Var. 2942 Var. 3027 Var. 3112 Var. 3197 Var. 3282 Var. 3367 110 ± 10  Var. 2773 Var. 2858 Var. 2943 Var. 3028 Var. 3113 Var. 3198 Var. 3283 Var. 3368 120 ± 10  Var. 2774 Var. 2859 Var. 2944 Var. 3029 Var. 3114 Var. 3199 Var. 3284 Var. 3369 125 ± 10  Var. 2775 Var. 2860 Var. 2945 Var. 3030 Var. 3115 Var. 3200 Var. 3285 Var. 3370 130 ± 10  Var. 2776 Var. 2861 Var. 2946 Var. 3031 Var. 3116 Var. 3201 Var. 3286 Var. 3371 140 ± 10  Var. 2777 Var. 2862 Var. 2947 Var. 3032 Var. 3117 Var. 3202 Var. 3287 Var. 3372 150 ± 10  Var. 2778 Var. 2863 Var. 2948 Var. 3033 Var. 3118 Var. 3203 Var. 3288 Var. 3373 160 ± 10  Var. 2779 Var. 2864 Var. 2949 Var. 3034 Var. 3119 Var. 3204 Var. 3289 Var. 3374 170 ± 10  Var. 2780 Var. 2865 Var. 2950 Var. 3035 Var. 3120 Var. 3205 Var. 3290 Var. 3375 175 ± 10  Var. 2781 Var. 2866 Var. 2951 Var. 3036 Var. 3121 Var. 3206 Var. 3291 Var. 3376 180 ± 10  Var. 2782 Var. 2867 Var. 2952 Var. 3037 Var. 3122 Var. 3207 Var. 3292 Var. 3377 190 ± 10  Var. 2783 Var. 2868 Var. 2953 Var. 3038 Var. 3123 Var. 3208 Var. 3293 Var. 3378 25 Var. 2784 Var. 2869 Var. 2954 Var. 3039 Var. 3124 Var. 3209 Var. 3294 Var. 3379 30 Var. 2785 Var. 2870 Var. 2955 Var. 3040 Var. 3125 Var. 3210 Var. 3295 Var. 3380 40 Var. 2786 Var. 2871 Var. 2956 Var. 3041 Var. 3126 Var. 3211 Var. 3296 Var. 3381 50 Var. 2787 Var. 2872 Var. 2957 Var. 3042 Var. 3127 Var. 3212 Var. 3297 Var. 3382 60 Var. 2788 Var. 2873 Var. 2958 Var. 3043 Var. 3128 Var. 3213 Var. 3298 Var. 3383 70 Var. 2789 Var. 2874 Var. 2959 Var. 3044 Var. 3129 Var. 3214 Var. 3299 Var. 3384 75 Var. 2790 Var. 2875 Var. 2960 Var. 3045 Var. 3130 Var. 3215 Var. 3300 Var. 3385 80 Var. 2791 Var. 2876 Var. 2961 Var. 3046 Var. 3131 Var. 3216 Var. 3301 Var. 3386 90 Var. 2792 Var. 2877 Var. 2962 Var. 3047 Var. 3132 Var. 3217 Var. 3302 Var. 3387 100 Var. 2793 Var. 2878 Var. 2963 Var. 3048 Var. 3133 Var. 3218 Var. 3303 Var. 3388 110 Var. 2794 Var. 2879 Var. 2964 Var. 3049 Var. 3134 Var. 3219 Var. 3304 Var. 3389 120 Var. 2795 Var. 2880 Var. 2965 Var. 3050 Var. 3135 Var. 3220 Var. 3305 Var. 3390 125 Var. 2796 Var. 2881 Var. 2966 Var. 3051 Var. 3136 Var. 3221 Var. 3306 Var. 3391 130 Var. 2797 Var. 2882 Var. 2967 Var. 3052 Var. 3137 Var. 3222 Var. 3307 Var. 3392 140 Var. 2798 Var. 2883 Var. 2968 Var. 3053 Var. 3138 Var. 3223 Var. 3308 Var. 3393 150 Var. 2799 Var. 2884 Var. 2969 Var. 3054 Var. 3139 Var. 3224 Var. 3309 Var. 3394 160 Var. 2800 Var. 2885 Var. 2970 Var. 3055 Var. 3140 Var. 3225 Var. 3310 Var. 3395 170 Var. 2801 Var. 2886 Var. 2971 Var. 3056 Var. 3141 Var. 3226 Var. 3311 Var. 3396 175 Var. 2802 Var. 2887 Var. 2972 Var. 3057 Var. 3142 Var. 3227 Var. 3312 Var. 3397 180 Var. 2803 Var. 2888 Var. 2973 Var. 3058 Var. 3143 Var. 3228 Var. 3313 Var. 3398 190 Var. 2804 Var. 2889 Var. 2974 Var. 3059 Var. 3144 Var. 3229 Var. 3314 Var. 3399 200 Var. 2805 Var. 2890 Var. 2975 Var. 3060 Var. 3145 Var. 3230 Var. 3315 Var. 3400 Var. = Variation

TABLE 6 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 8 ± 1% 9 ± 1% 10 ± 1% 11 ± 1% 12 ± 1% 13 ± 1% 14 ± 1% 15 ± 1% Non-Ionic  10-200 Var. 3401 Var. 3486 Var. 3571 Var. 3656 Var. 3741 Var. 3826 Var. 3911 Var. 3996 Surfactant  10-175 Var. 3402 Var. 3487 Var. 3572 Var. 3657 Var. 3742 Var. 3827 Var. 3912 Var. 3997 (ppm)  10-150 Var. 3403 Var. 3488 Var. 3573 Var. 3658 Var. 3743 Var. 3828 Var. 3913 Var. 3998  10-125 Var. 3404 Var. 3489 Var. 3574 Var. 3659 Var. 3744 Var. 3829 Var. 3914 Var. 3999  10-100 Var. 3405 Var. 3490 Var. 3575 Var. 3660 Var. 3745 Var. 3830 Var. 3915 Var. 4000 10-90 Var. 3406 Var. 3491 Var. 3576 Var. 3661 Var. 3746 Var. 3831 Var. 3916 Var. 4001 10-80 Var. 3407 Var. 3492 Var. 3577 Var. 3662 Var. 3747 Var. 3832 Var. 3917 Var. 4002 10-75 Var. 3408 Var. 3493 Var. 3578 Var. 3663 Var. 3748 Var. 3833 Var. 3918 Var. 4003 10-70 Var. 3409 Var. 3494 Var. 3579 Var. 3664 Var. 3749 Var. 3834 Var. 3919 Var. 4004 10-60 Var. 3410 Var. 3495 Var. 3580 Var. 3665 Var. 3750 Var. 3835 Var. 3920 Var. 4005 10-50 Var. 3411 Var. 3496 Var. 3581 Var. 3666 Var. 3751 Var. 3836 Var. 3921 Var. 4006 10-25 Var. 3412 Var. 3497 Var. 3582 Var. 3667 Var. 3752 Var. 3837 Var. 3922 Var. 4007  25-200 Var. 3413 Var. 3498 Var. 3583 Var. 3668 Var. 3753 Var. 3838 Var. 3923 Var. 4008  25-175 Var. 3414 Var. 3499 Var. 3584 Var. 3669 Var. 3754 Var. 3839 Var. 3924 Var. 4009  25-150 Var. 3415 Var. 3500 Var. 3585 Var. 3670 Var. 3755 Var. 3840 Var. 3925 Var. 4010  25-125 Var. 3416 Var. 3501 Var. 3586 Var. 3671 Var. 3756 Var. 3841 Var. 3926 Var. 4011  25-100 Var. 3417 Var. 3502 Var. 3587 Var. 3672 Var. 3757 Var. 3842 Var. 3927 Var. 4012 25-90 Var. 3418 Var. 3503 Var. 3588 Var. 3673 Var. 3758 Var. 3843 Var. 3928 Var. 4013 25-80 Var. 3419 Var. 3504 Var. 3589 Var. 3674 Var. 3759 Var. 3844 Var. 3929 Var. 4014 25-70 Var. 3420 Var. 3505 Var. 3590 Var. 3675 Var. 3760 Var. 3845 Var. 3930 Var. 4015 25-60 Var. 3421 Var. 3506 Var. 3591 Var. 3676 Var. 3761 Var. 3846 Var. 3931 Var. 4016 25-50 Var. 3422 Var. 3507 Var. 3592 Var. 3677 Var. 3762 Var. 3847 Var. 3932 Var. 4017  50-200 Var. 3423 Var. 3508 Var. 3593 Var. 3678 Var. 3763 Var. 3848 Var. 3933 Var. 4018  50-175 Var. 3424 Var. 3509 Var. 3594 Var. 3679 Var. 3764 Var. 3849 Var. 3934 Var. 4019  50-150 Var. 3425 Var. 3510 Var. 3595 Var. 3680 Var. 3765 Var. 3850 Var. 3935 Var. 4020  50-125 Var. 3426 Var. 3511 Var. 3596 Var. 3681 Var. 3766 Var. 3851 Var. 3936 Var. 4021 50-90 Var. 3427 Var. 3512 Var. 3597 Var. 3682 Var. 3767 Var. 3852 Var. 3937 Var. 4022 50-80 Var. 3428 Var. 3513 Var. 3598 Var. 3683 Var. 3768 Var. 3853 Var. 3938 Var. 4023  75-200 Var. 3429 Var. 3514 Var. 3599 Var. 3684 Var. 3769 Var. 3854 Var. 3939 Var. 4024  75-175 Var. 3430 Var. 3515 Var. 3600 Var. 3685 Var. 3770 Var. 3855 Var. 3940 Var. 4025  75-150 Var. 3431 Var. 3516 Var. 3601 Var. 3686 Var. 3771 Var. 3856 Var. 3941 Var. 4026 100-200 Var. 3432 Var. 3517 Var. 3602 Var. 3687 Var. 3772 Var. 3857 Var. 3942 Var. 4027 100-175 Var. 3433 Var. 3518 Var. 3603 Var. 3688 Var. 3773 Var. 3858 Var. 3943 Var. 4028 50 ± 25 Var. 3434 Var. 3519 Var. 3604 Var. 3689 Var. 3774 Var. 3859 Var. 3944 Var. 4029 60 ± 25 Var. 3435 Var. 3520 Var. 3605 Var. 3690 Var. 3775 Var. 3860 Var. 3945 Var. 4030 70 ± 25 Var. 3436 Var. 3521 Var. 3606 Var. 3691 Var. 3776 Var. 3861 Var. 3946 Var. 4031 75 ± 25 Var. 3437 Var. 3522 Var. 3607 Var. 3692 Var. 3777 Var. 3862 Var. 3947 Var. 4032 80 ± 25 Var. 3438 Var. 3523 Var. 3608 Var. 3693 Var. 3778 Var. 3863 Var. 3948 Var. 4033 90 ± 25 Var. 3439 Var. 3524 Var. 3609 Var. 3694 Var. 3779 Var. 3864 Var. 3949 Var. 4034 100 ± 25  Var. 3440 Var. 3525 Var. 3610 Var. 3695 Var. 3780 Var. 3865 Var. 3950 Var. 4035 125 ± 25  Var. 3441 Var. 3526 Var. 3611 Var. 3696 Var. 3781 Var. 3866 Var. 3951 Var. 4036 150 ± 25  Var. 3442 Var. 3527 Var. 3612 Var. 3697 Var. 3782 Var. 3867 Var. 3952 Var. 4037 175 ± 25  Var. 3443 Var. 3528 Var. 3613 Var. 3698 Var. 3783 Var. 3868 Var. 3953 Var. 4038 30 ± 10 Var. 3444 Var. 3529 Var. 3614 Var. 3699 Var. 3784 Var. 3869 Var. 3954 Var. 4039 40 ± 10 Var. 3445 Var. 3530 Var. 3615 Var. 3700 Var. 3785 Var. 3870 Var. 3955 Var. 4040 50 ± 10 Var. 3446 Var. 3531 Var. 3616 Var. 3701 Var. 3786 Var. 3871 Var. 3956 Var. 4041 60 ± 10 Var. 3447 Var. 3532 Var. 3617 Var. 3702 Var. 3787 Var. 3872 Var. 3957 Var. 4042 70 ± 10 Var. 3448 Var. 3533 Var. 3618 Var. 3703 Var. 3788 Var. 3873 Var. 3958 Var. 4043 75 ± 10 Var. 3449 Var. 3534 Var. 3619 Var. 3704 Var. 3789 Var. 3874 Var. 3959 Var. 4044 80 ± 10 Var. 3450 Var. 3535 Var. 3620 Var. 3705 Var. 3790 Var. 3875 Var. 3960 Var. 4045 90 ± 10 Var. 3451 Var. 3536 Var. 3621 Var. 3706 Var. 3791 Var. 3876 Var. 3961 Var. 4046 100 ± 10  Var. 3452 Var. 3537 Var. 3622 Var. 3707 Var. 3792 Var. 3877 Var. 3962 Var. 4047 110 ± 10  Var. 3453 Var. 3538 Var. 3623 Var. 3708 Var. 3793 Var. 3878 Var. 3963 Var. 4048 120 ± 10  Var. 3454 Var. 3539 Var. 3624 Var. 3709 Var. 3794 Var. 3879 Var. 3964 Var. 4049 125 ± 10  Var. 3455 Var. 3540 Var. 3625 Var. 3710 Var. 3795 Var. 3880 Var. 3965 Var. 4050 130 ± 10  Var. 3456 Var. 3541 Var. 3626 Var. 3711 Var. 3796 Var. 3881 Var. 3966 Var. 4051 140 ± 10  Var. 3457 Var. 3542 Var. 3627 Var. 3712 Var. 3797 Var. 3882 Var. 3967 Var. 4052 150 ± 10  Var. 3458 Var. 3543 Var. 3628 Var. 3713 Var. 3798 Var. 3883 Var. 3968 Var. 4053 160 ± 10  Var. 3459 Var. 3544 Var. 3629 Var. 3714 Var. 3799 Var. 3884 Var. 3969 Var. 4054 170 ± 10  Var. 3460 Var. 3545 Var. 3630 Var. 3715 Var. 3800 Var. 3885 Var. 3970 Var. 4055 175 ± 10  Var. 3461 Var. 3546 Var. 3631 Var. 3716 Var. 3801 Var. 3886 Var. 3971 Var. 4056 180 ± 10  Var. 3462 Var. 3547 Var. 3632 Var. 3717 Var. 3802 Var. 3887 Var. 3972 Var. 4057 190 ± 10  Var. 3463 Var. 3548 Var. 3633 Var. 3718 Var. 3803 Var. 3888 Var. 3973 Var. 4058 25 Var. 3464 Var. 3549 Var. 3634 Var. 3719 Var. 3804 Var. 3889 Var. 3974 Var. 4059 30 Var. 3465 Var. 3550 Var. 3635 Var. 3720 Var. 3805 Var. 3890 Var. 3975 Var. 4060 40 Var. 3466 Var. 3551 Var. 3636 Var. 3721 Var. 3806 Var. 3891 Var. 3976 Var. 4061 50 Var. 3467 Var. 3552 Var. 3637 Var. 3722 Var. 3807 Var. 3892 Var. 3977 Var. 4062 60 Var. 3468 Var. 3553 Var. 3638 Var. 3723 Var. 3808 Var. 3893 Var. 3978 Var. 4063 70 Var. 3469 Var. 3554 Var. 3639 Var. 3724 Var. 3809 Var. 3894 Var. 3979 Var. 4064 75 Var. 3470 Var. 3555 Var. 3640 Var. 3725 Var. 3810 Var. 3895 Var. 3980 Var. 4065 80 Var. 3471 Var. 3556 Var. 3641 Var. 3726 Var. 3811 Var. 3896 Var. 3981 Var. 4066 90 Var. 3472 Var. 3557 Var. 3642 Var. 3727 Var. 3812 Var. 3897 Var. 3982 Var. 4067 100 Var. 3473 Var. 3558 Var. 3643 Var. 3728 Var. 3813 Var. 3898 Var. 3983 Var. 4068 110 Var. 3474 Var. 3559 Var. 3644 Var. 3729 Var. 3814 Var. 3899 Var. 3984 Var. 4069 120 Var. 3475 Var. 3560 Var. 3645 Var. 3730 Var. 3815 Var. 3900 Var. 3985 Var. 4070 125 Var. 3476 Var. 3561 Var. 3646 Var. 3731 Var. 3816 Var. 3901 Var. 3986 Var. 4071 130 Var. 3477 Var. 3562 Var. 3647 Var. 3732 Var. 3817 Var. 3902 Var. 3987 Var. 4072 140 Var. 3478 Var. 3563 Var. 3648 Var. 3733 Var. 3818 Var. 3903 Var. 3988 Var. 4073 150 Var. 3479 Var. 3564 Var. 3649 Var. 3734 Var. 3819 Var. 3904 Var. 3989 Var. 4074 160 Var. 3480 Var. 3565 Var. 3650 Var. 3735 Var. 3820 Var. 3905 Var. 3990 Var. 4075 170 Var. 3481 Var. 3566 Var. 3651 Var. 3736 Var. 3821 Var. 3906 Var. 3991 Var. 4076 175 Var. 3482 Var. 3567 Var. 3652 Var. 3737 Var. 3822 Var. 3907 Var. 3992 Var. 4077 180 Var. 3483 Var. 3568 Var. 3653 Var. 3738 Var. 3823 Var. 3908 Var. 3993 Var. 4078 190 Var. 3484 Var. 3569 Var. 3654 Var. 3739 Var. 3824 Var. 3909 Var. 3994 Var. 4079 200 Var. 3485 Var. 3570 Var. 3655 Var. 3740 Var. 3825 Var. 3910 Var. 3995 Var. 4080 Var. = Variation

TABLE 7 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 16 ± 1% 17 ± 1% 18 ± 1% 19 ± 1% 2% 3% 4% 5% Non-Ionic  10-200 Var. 4081 Var. 4166 Var. 4251 Var. 4336 Var. 4421 Var. 4506 Var. 4591 Var. 4676 Surfactant  10-175 Var. 4082 Var. 4167 Var. 4252 Var. 4337 Var. 4422 Var. 4507 Var. 4592 Var. 4677 (ppm)  10-150 Var. 4083 Var. 4168 Var. 4253 Var. 4338 Var. 4423 Var. 4508 Var. 4593 Var. 4678  10-125 Var. 4084 Var. 4169 Var. 4254 Var. 4339 Var. 4424 Var. 4509 Var. 4594 Var. 4679  10-100 Var. 4085 Var. 4170 Var. 4255 Var. 4340 Var. 4425 Var. 4510 Var. 4595 Var. 4680 10-90 Var. 4086 Var. 4171 Var. 4256 Var. 4341 Var. 4426 Var. 4511 Var. 4596 Var. 4681 10-80 Var. 4087 Var. 4172 Var. 4257 Var. 4342 Var. 4427 Var. 4512 Var. 4597 Var. 4682 10-75 Var. 4088 Var. 4173 Var. 4258 Var. 4343 Var. 4428 Var. 4513 Var. 4598 Var. 4683 10-70 Var. 4089 Var. 4174 Var. 4259 Var. 4344 Var. 4429 Var. 4514 Var. 4599 Var. 4684 10-60 Var. 4090 Var. 4175 Var. 4260 Var. 4345 Var. 4430 Var. 4515 Var. 4600 Var. 4685 10-50 Var. 4091 Var. 4176 Var. 4261 Var. 4346 Var. 4431 Var. 4516 Var. 4601 Var. 4686 10-25 Var. 4092 Var. 4177 Var. 4262 Var. 4347 Var. 4432 Var. 4517 Var. 4602 Var. 4687  25-200 Var. 4093 Var. 4178 Var. 4263 Var. 4348 Var. 4433 Var. 4518 Var. 4603 Var. 4688  25-175 Var. 4094 Var. 4179 Var. 4264 Var. 4349 Var. 4434 Var. 4519 Var. 4604 Var. 4689  25-150 Var. 4095 Var. 4180 Var. 4265 Var. 4350 Var. 4435 Var. 4520 Var. 4605 Var. 4690  25-125 Var. 4096 Var. 4181 Var. 4266 Var. 4351 Var. 4436 Var. 4521 Var. 4606 Var. 4691  25-100 Var. 4097 Var. 4182 Var. 4267 Var. 4352 Var. 4437 Var. 4522 Var. 4607 Var. 4692 25-90 Var. 4098 Var. 4183 Var. 4268 Var. 4353 Var. 4438 Var. 4523 Var. 4608 Var. 4693 25-80 Var. 4099 Var. 4184 Var. 4269 Var. 4354 Var. 4439 Var. 4524 Var. 4609 Var. 4694 25-70 Var. 4100 Var. 4185 Var. 4270 Var. 4355 Var. 4440 Var. 4525 Var. 4610 Var. 4695 25-60 Var. 4101 Var. 4186 Var. 4271 Var. 4356 Var. 4441 Var. 4526 Var. 4611 Var. 4696 25-50 Var. 4102 Var. 4187 Var. 4272 Var. 4357 Var. 4442 Var. 4527 Var. 4612 Var. 4697  50-200 Var. 4103 Var. 4188 Var. 4273 Var. 4358 Var. 4443 Var. 4528 Var. 4613 Var. 4698  50-175 Var. 4104 Var. 4189 Var. 4274 Var. 4359 Var. 4444 Var. 4529 Var. 4614 Var. 4699  50-150 Var. 4105 Var. 4190 Var. 4275 Var. 4360 Var. 4445 Var. 4530 Var. 4615 Var. 4700  50-125 Var. 4106 Var. 4191 Var. 4276 Var. 4361 Var. 4446 Var. 4531 Var. 4616 Var. 4701 50-90 Var. 4107 Var. 4192 Var. 4277 Var. 4362 Var. 4447 Var. 4532 Var. 4617 Var. 4702 50-80 Var. 4108 Var. 4193 Var. 4278 Var. 4363 Var. 4448 Var. 4533 Var. 4618 Var. 4703  75-200 Var. 4109 Var. 4194 Var. 4279 Var. 4364 Var. 4449 Var. 4534 Var. 4619 Var. 4704  75-175 Var. 4110 Var. 4195 Var. 4280 Var. 4365 Var. 4450 Var. 4535 Var. 4620 Var. 4705  75-150 Var. 4111 Var. 4196 Var. 4281 Var. 4366 Var. 4451 Var. 4536 Var. 4621 Var. 4706 100-200 Var. 4112 Var. 4197 Var. 4282 Var. 4367 Var. 4452 Var. 4537 Var. 4622 Var. 4707 100-175 Var. 4113 Var. 4198 Var. 4283 Var. 4368 Var. 4453 Var. 4538 Var. 4623 Var. 4708 50 ± 25 Var. 4114 Var. 4199 Var. 4284 Var. 4369 Var. 4454 Var. 4539 Var. 4624 Var. 4709 60 ± 25 Var. 4115 Var. 4200 Var. 4285 Var. 4370 Var. 4455 Var. 4540 Var. 4625 Var. 4710 70 ± 25 Var. 4116 Var. 4201 Var. 4286 Var. 4371 Var. 4456 Var. 4541 Var. 4626 Var. 4711 75 ± 25 Var. 4117 Var. 4202 Var. 4287 Var. 4372 Var. 4457 Var. 4542 Var. 4627 Var. 4712 80 ± 25 Var. 4118 Var. 4203 Var. 4288 Var. 4373 Var. 4458 Var. 4543 Var. 4628 Var. 4713 90 ± 25 Var. 4119 Var. 4204 Var. 4289 Var. 4374 Var. 4459 Var. 4544 Var. 4629 Var. 4714 100 ± 25  Var. 4120 Var. 4205 Var. 4290 Var. 4375 Var. 4460 Var. 4545 Var. 4630 Var. 4715 125 ± 25  Var. 4121 Var. 4206 Var. 4291 Var. 4376 Var. 4461 Var. 4546 Var. 4631 Var. 4716 150 ± 25  Var. 4122 Var. 4207 Var. 4292 Var. 4377 Var. 4462 Var. 4547 Var. 4632 Var. 4717 175 ± 25  Var. 4123 Var. 4208 Var. 4293 Var. 4378 Var. 4463 Var. 4548 Var. 4633 Var. 4718 30 ± 10 Var. 4124 Var. 4209 Var. 4294 Var. 4379 Var. 4464 Var. 4549 Var. 4634 Var. 4719 40 ± 10 Var. 4125 Var. 4210 Var. 4295 Var. 4380 Var. 4465 Var. 4550 Var. 4635 Var. 4720 50 ± 10 Var. 4126 Var. 4211 Var. 4296 Var. 4381 Var. 4466 Var. 4551 Var. 4636 Var. 4721 60 ± 10 Var. 4127 Var. 4212 Var. 4297 Var. 4382 Var. 4467 Var. 4552 Var. 4637 Var. 4722 70 ± 10 Var. 4128 Var. 4213 Var. 4298 Var. 4383 Var. 4468 Var. 4553 Var. 4638 Var. 4723 75 ± 10 Var. 4129 Var. 4214 Var. 4299 Var. 4384 Var. 4469 Var. 4554 Var. 4639 Var. 4724 80 ± 10 Var. 4130 Var. 4215 Var. 4300 Var. 4385 Var. 4470 Var. 4555 Var. 4640 Var. 4725 90 ± 10 Var. 4131 Var. 4216 Var. 4301 Var. 4386 Var. 4471 Var. 4556 Var. 4641 Var. 4726 100 ± 10  Var. 4132 Var. 4217 Var. 4302 Var. 4387 Var. 4472 Var. 4557 Var. 4642 Var. 4727 110 ± 10  Var. 4133 Var. 4218 Var. 4303 Var. 4338 Var. 4473 Var. 4558 Var. 4643 Var. 4728 120-10  Var. 4134 Var. 4219 Var. 4304 Var. 4389 Var. 4474 Var. 4559 Var. 4644 Var. 4729 125 ± 10  Var. 4135 Var. 4220 Var. 4305 Var. 4390 Var. 4475 Var. 4560 Var. 4645 Var. 4730 130 ± 10  Var. 4136 Var. 4221 Var. 4306 Var. 4391 Var. 4476 Var. 4561 Var. 4646 Var. 4731 140 ± 10  Var. 4137 Var. 4222 Var. 4307 Var. 4392 Var. 4477 Var. 4562 Var. 4647 Var. 4732 150 ± 10  Var. 4138 Var. 4223 Var. 4308 Var. 4393 Var. 4478 Var. 4563 Var. 4648 Var. 4733 160 ± 10  Var. 4139 Var. 4224 Var. 4309 Var. 4394 Var. 4479 Var. 4564 Var. 4649 Var. 4734 170 ± 10  Var. 4140 Var. 4225 Var. 4310 Var. 4395 Var. 4480 Var. 4565 Var. 4650 Var. 4735 175 ± 10  Var. 4141 Var. 4226 Var. 4311 Var. 4396 Var. 4481 Var. 4566 Var. 4651 Var. 4736 180 ± 10  Var. 4142 Var. 4227 Var. 4312 Var. 4397 Var. 4482 Var. 4567 Var. 4652 Var. 4737 190 ± 10  Var. 4143 Var. 4228 Var. 4313 Var. 4398 Var. 4483 Var. 4568 Var. 4653 Var. 4738 25 Var. 4144 Var. 4229 Var. 4314 Var. 4399 Var. 4484 Var. 4569 Var. 4654 Var. 4739 30 Var. 4145 Var. 4230 Var. 4315 Var. 4400 Var. 4485 Var. 4570 Var. 4655 Var. 4740 40 Var. 4146 Var. 4231 Var. 4316 Var. 4401 Var. 4486 Var. 4571 Var. 4656 Var. 4741 50 Var. 4147 Var. 4232 Var. 4317 Var. 4402 Var. 4487 Var. 4572 Var. 4657 Var. 4742 60 Var. 4148 Var. 4233 Var. 4318 Var. 4403 Var. 4488 Var. 4573 Var. 4658 Var. 4743 70 Var. 4149 Var. 4234 Var. 4319 Var. 4404 Var. 4489 Var. 4574 Var. 4659 Var. 4744 75 Var. 4150 Var. 4235 Var. 4320 Var. 4405 Var. 4490 Var. 4575 Var. 4660 Var. 4745 80 Var. 4151 Var. 4236 Var. 4321 Var. 4406 Var. 4491 Var. 4576 Var. 4661 Var. 4746 90 Var. 4152 Var. 4237 Var. 4322 Var. 4407 Var. 4492 Var. 4577 Var. 4662 Var. 4747 100 Var. 4153 Var. 4238 Var. 4323 Var. 4408 Var. 4493 Var. 4578 Var. 4663 Var. 4748 110 Var. 4154 Var. 4239 Var. 4324 Var. 4409 Var. 4494 Var. 4579 Var. 4664 Var. 4749 120 Var. 4155 Var. 4240 Var. 4325 Var. 4410 Var. 4495 Var. 4580 Var. 4665 Var. 4750 125 Var. 4156 Var. 4241 Var. 4326 Var. 4411 Var. 4496 Var. 4581 Var. 4666 Var. 4751 130 Var. 4157 Var. 4242 Var. 4327 Var. 4412 Var. 4497 Var. 4582 Var. 4667 Var. 4752 140 Var. 4158 Var. 4243 Var. 4328 Var. 4413 Var. 4498 Var. 4583 Var. 4668 Var. 4753 150 Var. 4159 Var. 4244 Var. 4329 Var. 4414 Var. 4499 Var. 4584 Var. 4669 Var. 4754 160 Var. 4160 Var. 4245 Var. 4330 Var. 4415 Var. 4500 Var. 4585 Var. 4670 Var. 4755 170 Var. 4161 Var. 4246 Var. 4331 Var. 4416 Var. 4501 Var. 4586 Var. 4671 Var. 4756 175 Var. 4162 Var. 4247 Var. 4332 Var. 4417 Var. 4502 Var. 4587 Var. 4672 Var. 4757 180 Var. 4163 Var. 4248 Var. 4333 Var. 4418 Var. 4503 Var. 4588 Var. 4673 Var. 4758 190 Var. 4164 Var. 4249 Var. 4334 Var. 4419 Var. 4504 Var. 4589 Var. 4674 Var. 4759 200 Var. 4165 Var. 4250 Var. 4335 Var. 4420 Var. 4505 Var. 4590 Var. 4675 Var. 4760 Var. = Variation

TABLE 8 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 6% 7% 8% 9% 10% 11% 12% 13% Non-Ionic  10-200 Var. 4761 Var. 4846 Var. 4931 Var. 5016 Var. 5101 Var. 5186 Var. 5271 Var. 5356 Surfactant  10-175 Var. 4762 Var. 4847 Var. 4932 Var. 5017 Var. 5102 Var. 5187 Var. 5272 Var. 5357 (ppm)  10-150 Var. 4763 Var. 4848 Var. 4933 Var. 5018 Var. 5103 Var. 5188 Var. 5273 Var. 5358  10-125 Var. 4764 Var. 4849 Var. 4934 Var. 5019 Var. 5104 Var. 5189 Var. 5274 Var. 5359  10-100 Var. 4765 Var. 4850 Var. 4935 Var. 5020 Var. 5105 Var. 5190 Var. 5275 Var. 5360 10-90 Var. 4766 Var. 4851 Var. 4936 Var. 5021 Var. 5106 Var. 5191 Var. 5276 Var. 5361 10-80 Var. 4767 Var. 4852 Var. 4937 Var. 5022 Var. 5107 Var. 5192 Var. 5277 Var. 5362 10-75 Var. 4768 Var. 4853 Var. 4938 Var. 5023 Var. 5108 Var. 5193 Var. 5278 Var. 5363 10-70 Var. 4769 Var. 4854 Var. 4939 Var. 5024 Var. 5109 Var. 5194 Var. 5279 Var. 5364 10-60 Var. 4770 Var. 4855 Var. 4940 Var. 5025 Var. 5110 Var. 5195 Var. 5280 Var. 5365 10-50 Var. 4771 Var. 4856 Var. 4941 Var. 5026 Var. 5111 Var. 5196 Var. 5281 Var. 5366 10-25 Var. 4772 Var. 4857 Var. 4942 Var. 5027 Var. 5112 Var. 5197 Var. 5282 Var. 5367  25-200 Var. 4773 Var. 4858 Var. 4943 Var. 5028 Var. 5113 Var. 5198 Var. 5283 Var. 5368  25-175 Var. 4774 Var. 4859 Var. 4944 Var. 5029 Var. 5114 Var. 5199 Var. 5284 Var. 5369  25-150 Var. 4775 Var. 4860 Var. 4945 Var. 5030 Var. 5115 Var. 5200 Var. 5285 Var. 5370  25-125 Var. 4776 Var. 4861 Var. 4946 Var. 5031 Var. 5116 Var. 5201 Var. 5286 Var. 5371  25-100 Var. 4777 Var. 4862 Var. 4947 Var. 5032 Var. 5117 Var. 5202 Var. 5287 Var. 5372 25-90 Var. 4778 Var. 4863 Var. 4948 Var. 5033 Var. 5118 Var. 5203 Var. 5288 Var. 5373 25-80 Var. 4779 Var. 4864 Var. 4949 Var. 5034 Var. 5119 Var. 5204 Var. 5289 Var. 5374 25-70 Var. 4780 Var. 4865 Var. 4950 Var. 5035 Var. 5120 Var. 5205 Var. 5290 Var. 5375 25-60 Var. 4781 Var. 4866 Var. 4951 Var. 5036 Var. 5121 Var. 5206 Var. 5291 Var. 5376 25-50 Var. 4782 Var. 4867 Var. 4952 Var. 5037 Var. 5122 Var. 5207 Var. 5292 Var. 5377  50-200 Var. 4783 Var. 4868 Var. 4953 Var. 5038 Var. 5123 Var. 5208 Var. 5293 Var. 5378  50-175 Var. 4784 Var. 4869 Var. 4954 Var. 5039 Var. 5124 Var. 5209 Var. 5294 Var. 5379  50-150 Var. 4785 Var. 4870 Var. 4955 Var. 5040 Var. 5125 Var. 5210 Var. 5295 Var. 5380  50-125 Var. 4786 Var. 4871 Var. 4956 Var. 5041 Var. 5126 Var. 5211 Var. 5296 Var. 5381 50-90 Var. 4787 Var. 4872 Var. 4957 Var. 5042 Var. 5127 Var. 5212 Var. 5297 Var. 5382 50-80 Var. 4788 Var. 4873 Var. 4958 Var. 5043 Var. 5128 Var. 5213 Var. 5298 Var. 5383  75-200 Var. 4789 Var. 4874 Var. 4959 Var. 5044 Var. 5129 Var. 5214 Var. 5299 Var. 5384  75-175 Var. 4790 Var. 4875 Var. 4960 Var. 5045 Var. 5130 Var. 5215 Var. 5300 Var. 5385  75-150 Var. 4791 Var. 4876 Var. 4961 Var. 5046 Var. 5131 Var. 5216 Var. 5301 Var. 5386 100-200 Var. 4792 Var. 4877 Var. 4962 Var. 5047 Var. 5132 Var. 5217 Var. 5302 Var. 5387 100-175 Var. 4793 Var. 4878 Var. 4963 Var. 5048 Var. 5133 Var. 5218 Var. 5303 Var. 5388 50 ± 25 Var. 4794 Var. 4879 Var. 4964 Var. 5049 Var. 5134 Var. 5219 Var. 5304 Var. 5389 60 ± 25 Var. 4795 Var. 4880 Var. 4965 Var. 5050 Var. 5135 Var. 5220 Var. 5305 Var. 5390 70 ± 25 Var. 4796 Var. 4881 Var. 4966 Var. 5051 Var. 5136 Var. 5221 Var. 5306 Var. 5391 75 ± 25 Var. 4797 Var. 4882 Var. 4967 Var. 5052 Var. 5137 Var. 5222 Var. 5307 Var. 5392 80 ± 25 Var. 4798 Var. 4883 Var. 4968 Var. 5053 Var. 5138 Var. 5223 Var. 5308 Var. 5393 90 ± 25 Var. 4799 Var. 4884 Var. 4969 Var. 5054 Var. 5139 Var. 5224 Var. 5309 Var. 5394 100 ± 25  Var. 4800 Var. 4885 Var. 4970 Var. 5055 Var. 5140 Var. 5225 Var. 5310 Var. 5395 125 ± 25  Var. 4801 Var. 4886 Var. 4971 Var. 5056 Var. 5141 Var. 5226 Var. 5311 Var. 5396 150 ± 25  Var. 4802 Var. 4887 Var. 4972 Var. 5057 Var. 5142 Var. 5227 Var. 5312 Var. 5397 175 ± 25  Var. 4803 Var. 4888 Var. 4973 Var. 5058 Var. 5143 Var. 5228 Var. 5313 Var. 5398 30 ± 10 Var. 4804 Var. 4889 Var. 4974 Var. 5059 Var. 5144 Var. 5229 Var. 5314 Var. 5399 40 ± 10 Var. 4805 Var. 4890 Var. 4975 Var. 5060 Var. 5145 Var. 5230 Var. 5315 Var. 5400 50 ± 10 Var. 4806 Var. 4891 Var. 4976 Var. 5061 Var. 5146 Var. 5231 Var. 5316 Var. 5401 60 ± 10 Var. 4807 Var. 4892 Var. 4977 Var. 5062 Var. 5147 Var. 5232 Var. 5317 Var. 5402 70 ± 10 Var. 4808 Var. 4893 Var. 4978 Var. 5063 Var. 5148 Var. 5233 Var. 5318 Var. 5403 75 ± 10 Var. 4809 Var. 4894 Var. 4979 Var. 5064 Var. 5149 Var. 5234 Var. 5319 Var. 5404 80 ± 10 Var. 4810 Var. 4895 Var. 4980 Var. 5065 Var. 5150 Var. 5235 Var. 5320 Var. 5405 90 ± 10 Var. 4811 Var. 4896 Var. 4981 Var. 5066 Var. 5151 Var. 5236 Var. 5321 Var. 5406 100 ± 10  Var. 4812 Var. 4897 Var. 4982 Var. 5067 Var. 5152 Var. 5237 Var. 5322 Var. 5407 110 ± 10  Var. 4813 Var. 4898 Var. 4983 Var. 5068 Var. 5153 Var. 5238 Var. 5323 Var. 5408 120 ± 10  Var. 4814 Var. 4899 Var. 4984 Var. 5069 Var. 5154 Var. 5239 Var. 5324 Var. 5409 125 ± 10  Var. 4815 Var. 4900 Var. 4985 Var. 5070 Var. 5155 Var. 5240 Var. 5325 Var. 5410 130 ± 10  Var. 4816 Var. 4901 Var. 4986 Var. 5071 Var. 5156 Var. 5241 Var. 5326 Var. 5411 140 ± 10  Var. 4817 Var. 4902 Var. 4987 Var. 5072 Var. 5157 Var. 5242 Var. 5327 Var. 5412 150 ± 10  Var. 4818 Var. 4903 Var. 4988 Var. 5073 Var. 5158 Var. 5243 Var. 5328 Var. 5413 160 ± 10  Var. 4819 Var. 4904 Var. 4989 Var. 5074 Var. 5159 Var. 5244 Var. 5329 Var. 5414 170 ± 10  Var. 4820 Var. 4905 Var. 4990 Var. 5075 Var. 5160 Var. 5245 Var. 5330 Var. 5415 175 ± 10  Var. 4821 Var. 4906 Var. 4991 Var. 5076 Var. 5161 Var. 5246 Var. 5331 Var. 5416 180 ± 10  Var. 4822 Var. 4907 Var. 4992 Var. 5077 Var. 5162 Var. 5247 Var. 5332 Var. 5417 190 ± 10  Var. 4823 Var. 4908 Var. 4993 Var. 5078 Var. 5163 Var. 5248 Var. 5333 Var. 5418 25 Var. 4824 Var. 4909 Var. 4994 Var. 5079 Var. 5164 Var. 5249 Var. 5334 Var. 5419 30 Var. 4825 Var. 4910 Var. 4995 Var. 5080 Var. 5165 Var. 5250 Var. 5335 Var. 5420 40 Var. 4826 Var. 4911 Var. 4996 Var. 5081 Var. 5166 Var. 5251 Var. 5336 Var. 5421 50 Var. 4827 Var. 4912 Var. 4997 Var. 5082 Var. 5167 Var. 5252 Var. 5337 Var. 5422 60 Var. 4828 Var. 4913 Var. 4998 Var. 5083 Var. 5168 Var. 5253 Var. 5338 Var. 5423 70 Var. 4829 Var. 4914 Var. 4999 Var. 5084 Var. 5169 Var. 5254 Var. 5339 Var. 5424 75 Var. 4830 Var. 4915 Var. 5000 Var. 5085 Var. 5170 Var. 5255 Var. 5340 Var. 5425 80 Var. 4831 Var. 4916 Var. 5001 Var. 5086 Var. 5171 Var. 5256 Var. 5341 Var. 5426 90 Var. 4832 Var. 4917 Var. 5002 Var. 5087 Var. 5172 Var. 5257 Var. 5342 Var. 5427 100 Var. 4833 Var. 4918 Var. 5003 Var. 5088 Var. 5173 Var. 5258 Var. 5343 Var. 5428 110 Var. 4834 Var. 4919 Var. 5004 Var. 5089 Var. 5174 Var. 5259 Var. 5344 Var. 5429 120 Var. 4835 Var. 4920 Var. 5005 Var. 5090 Var. 5175 Var. 5260 Var. 5345 Var. 5430 125 Var. 4836 Var. 4921 Var. 5006 Var. 5091 Var. 5176 Var. 5261 Var. 5346 Var. 5431 130 Var. 4837 Var. 4922 Var. 5007 Var. 5092 Var. 5177 Var. 5262 Var. 5347 Var. 5432 140 Var. 4838 Var. 4923 Var. 5008 Var. 5093 Var. 5178 Var. 5263 Var. 5348 Var. 5433 150 Var. 4839 Var. 4924 Var. 5009 Var. 5094 Var. 5179 Var. 5264 Var. 5349 Var. 5434 160 Var. 4840 Var. 4925 Var. 5010 Var. 5095 Var. 5180 Var. 5265 Var. 5350 Var. 5435 170 Var. 4841 Var. 4926 Var. 5011 Var. 5096 Var. 5181 Var. 5266 Var. 5351 Var. 5436 175 Var. 4842 Var. 4927 Var. 5012 Var. 5097 Var. 5182 Var. 5267 Var. 5352 Var. 5437 180 Var. 4843 Var. 4928 Var. 5013 Var. 5098 Var. 5183 Var. 5268 Var. 5353 Var. 5438 190 Var. 4844 Var. 4929 Var. 5014 Var. 5099 Var. 5184 Var. 5269 Var. 5354 Var. 5439 200 Var. 4845 Var. 4930 Var. 5015 Var. 5100 Var. 5185 Var. 5270 Var. 5355 Var. 5440 Var. = Variation

TABLE 9 Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions. Sugar or Sugar Alcohol (%) 14% 15% 16% 17% 18% 19% 20% Non-Ionic  10-200 Var. 5441 Var. 5526 Var. 5611 Var. 5696 Var. 5781 Var. 5866 Var. 5951 Surfactant  10-175 Var. 5442 Var. 5527 Var. 5612 Var. 5697 Var. 5782 Var. 5867 Var. 5952 (ppm)  10-150 Var. 5443 Var. 5528 Var. 5613 Var. 5698 Var. 5783 Var. 5868 Var. 5953  10-125 Var. 5444 Var. 5529 Var. 5614 Var. 5699 Var. 5784 Var. 5869 Var. 5954  10-100 Var. 5445 Var. 5530 Var. 5615 Var. 5700 Var. 5785 Var. 5870 Var. 5955 10-90 Var. 5446 Var. 5531 Var. 5616 Var. 5701 Var. 5786 Var. 5871 Var. 5956 10-80 Var. 5447 Var. 5532 Var. 5617 Var. 5702 Var. 5787 Var. 5872 Var. 5957 10-75 Var. 5448 Var. 5533 Var. 5618 Var. 5703 Var. 5788 Var. 5873 Var. 5958 10-70 Var. 5449 Var. 5534 Var. 5619 Var. 5704 Var. 5789 Var. 5874 Var. 5959 10-60 Var. 5450 Var. 5535 Var. 5620 Var. 5705 Var. 5790 Var. 5875 Var. 5960 10-50 Var. 5451 Var. 5536 Var. 5621 Var. 5706 Var. 5791 Var. 5876 Var. 5961 10-25 Var. 5452 Var. 5537 Var. 5622 Var. 5707 Var. 5792 Var. 5877 Var. 5962  25-200 Var. 5453 Var. 5538 Var. 5623 Var. 5708 Var. 5793 Var. 5878 Var. 5963  25-175 Var. 5454 Var. 5539 Var. 5624 Var. 5709 Var. 5794 Var. 5879 Var. 5964  25-150 Var. 5455 Var. 5540 Var. 5625 Var. 5710 Var. 5795 Var. 5880 Var. 5965  25-125 Var. 5456 Var. 5541 Var. 5626 Var. 5711 Var. 5796 Var. 5881 Var. 5966  25-100 Var. 5457 Var. 5542 Var. 5627 Var. 5712 Var. 5797 Var. 5882 Var. 5967 25-90 Var. 5458 Var. 5543 Var. 5628 Var. 5713 Var. 5798 Var. 5883 Var. 5968 25-80 Var. 5459 Var. 5544 Var. 5629 Var. 5714 Var. 5799 Var. 5884 Var. 5969 25-70 Var. 5460 Var. 5545 Var. 5630 Var. 5715 Var. 5800 Var. 5885 Var. 5970 25-60 Var. 5461 Var. 5546 Var. 5631 Var. 5716 Var. 5801 Var. 5886 Var. 5971 25-50 Var. 5462 Var. 5547 Var. 5632 Var. 5717 Var. 5802 Var. 5887 Var. 5972  50-200 Var. 5463 Var. 5548 Var. 5633 Var. 5718 Var. 5803 Var. 5888 Var. 5973  50-175 Var. 5464 Var. 5549 Var. 5634 Var. 5719 Var. 5804 Var. 5889 Var. 5974  50-150 Var. 5465 Var. 5550 Var. 5635 Var. 5720 Var. 5805 Var. 5890 Var. 5975  50-125 Var. 5466 Var. 5551 Var. 5636 Var. 5721 Var. 5806 Var. 5891 Var. 5976 50-90 Var. 5467 Var. 5552 Var. 5637 Var. 5722 Var. 5807 Var. 5892 Var. 5977 50-80 Var. 5468 Var. 5553 Var. 5638 Var. 5723 Var. 5808 Var. 5893 Var. 5978  75-200 Var. 5469 Var. 5554 Var. 5639 Var. 5724 Var. 5809 Var. 5894 Var. 5979  75-175 Var. 5470 Var. 5555 Var. 5640 Var. 5725 Var. 5810 Var. 5895 Var. 5980  75-150 Var. 5471 Var. 5556 Var. 5641 Var. 5726 Var. 5811 Var. 5896 Var. 5981 100-200 Var. 5472 Var. 5557 Var. 5642 Var. 5727 Var. 5812 Var. 5897 Var. 5982 100-175 Var. 5473 Var. 5558 Var. 5643 Var. 5728 Var. 5813 Var. 5898 Var. 5983 50 ± 25 Var. 5474 Var. 5559 Var. 5644 Var. 5729 Var. 5814 Var. 5899 Var. 5984 60 ± 25 Var. 5475 Var. 5560 Var. 5645 Var. 5730 Var. 5815 Var. 5900 Var. 5985 70 ± 25 Var. 5476 Var. 5561 Var. 5646 Var. 5731 Var. 5816 Var. 5901 Var. 5986 75 ± 25 Var. 5477 Var. 5562 Var. 5647 Var. 5732 Var. 5817 Var. 5902 Var. 5987 80 ± 25 Var. 5478 Var. 5563 Var. 5648 Var. 5733 Var. 5818 Var. 5903 Var. 5988 90 ± 25 Var. 5479 Var. 5564 Var. 5649 Var. 5734 Var. 5819 Var. 5904 Var. 5989 100 ± 25  Var. 5480 Var. 5565 Var. 5650 Var. 5735 Var. 5820 Var. 5905 Var. 5990 125 ± 25  Var. 5481 Var. 5566 Var. 5651 Var. 5736 Var. 5821 Var. 5906 Var. 5991 150 ± 25  Var. 5482 Var. 5567 Var. 5652 Var. 5737 Var. 5822 Var. 5907 Var. 5992 175 ± 25  Var. 5483 Var. 5568 Var. 5653 Var. 5738 Var. 5823 Var. 5908 Var. 5993 30 ± 10 Var. 5484 Var. 5569 Var. 5654 Var. 5739 Var. 5824 Var. 5909 Var. 5994 40 ± 10 Var. 5485 Var. 5570 Var. 5655 Var. 5740 Var. 5825 Var. 5910 Var. 5995 50 ± 10 Var. 5486 Var. 5571 Var. 5656 Var. 5741 Var. 5826 Var. 5911 Var. 5996 60 ± 10 Var. 5487 Var. 5572 Var. 5657 Var. 5742 Var. 5827 Var. 5912 Var. 5997 70 ± 10 Var. 5488 Var. 5573 Var. 5658 Var. 5743 Var. 5828 Var. 5913 Var. 5998 75 ± 10 Var. 5489 Var. 5574 Var. 5659 Var. 5744 Var. 5829 Var. 5914 Var. 5999 80 ± 10 Var. 5490 Var. 5575 Var. 5660 Var. 5745 Var. 5830 Var. 5915 Var. 6000 90 ± 10 Var. 5491 Var. 5576 Var. 5661 Var. 5746 Var. 5831 Var. 5916 Var. 6001 100 ± 10  Var. 5492 Var. 5577 Var. 5662 Var. 5747 Var. 5832 Var. 5917 Var. 6002 110 ± 10  Var. 5493 Var. 5578 Var. 5663 Var. 5748 Var. 5833 Var. 5918 Var. 6003 120 ± 10  Var. 5494 Var. 5579 Var. 5664 Var. 5749 Var. 5834 Var. 5919 Var. 6004 125 ± 10  Var. 5495 Var. 5580 Var. 5665 Var. 5750 Var. 5835 Var. 5920 Var. 6005 130 ± 10  Var. 5496 Var. 5581 Var. 5666 Var. 5751 Var. 5836 Var. 5921 Var. 6006 140 ± 10  Var. 5497 Var. 5582 Var. 5667 Var. 5752 Var. 5837 Var. 5922 Var. 6007 150 ± 10  Var. 5498 Var. 5583 Var. 5668 Var. 5753 Var. 5838 Var. 5923 Var. 6008 160 ± 10  Var. 5499 Var. 5584 Var. 5669 Var. 5754 Var. 5839 Var. 5924 Var. 6009 170 ± 10  Var. 5500 Var. 5585 Var. 5670 Var. 5755 Var. 5840 Var. 5925 Var. 6010 175 ± 10  Var. 5501 Var. 5586 Var. 5671 Var. 5756 Var. 5841 Var. 5926 Var. 6011 180 ± 10  Var. 5502 Var. 5587 Var. 5672 Var. 5757 Var. 5842 Var. 5927 Var. 6012 190 ± 10  Var. 5503 Var. 5588 Var. 5673 Var. 5758 Var. 5843 Var. 5928 Var. 6013 25 Var. 5504 Var. 5589 Var. 5674 Var. 5759 Var. 5844 Var. 5929 Var. 6014 30 Var. 5505 Var. 5590 Var. 5675 Var. 5760 Var. 5845 Var. 5930 Var. 6015 40 Var. 5506 Var. 5591 Var. 5676 Var. 5761 Var. 5846 Var. 5931 Var. 6016 50 Var. 5507 Var. 5592 Var. 5677 Var. 5762 Var. 5847 Var. 5932 Var. 6017 60 Var. 5508 Var. 5593 Var. 5678 Var. 5763 Var. 5848 Var. 5933 Var. 6018 70 Var. 5509 Var. 5594 Var. 5679 Var. 5764 Var. 5849 Var. 5934 Var. 6019 75 Var. 5510 Var. 5595 Var. 5680 Var. 5765 Var. 5850 Var. 5935 Var. 6020 80 Var. 5511 Var. 5596 Var. 5681 Var. 5766 Var. 5851 Var. 5936 Var. 6021 90 Var. 5512 Var. 5597 Var. 5682 Var. 5767 Var. 5852 Var. 5937 Var. 6022 100 Var. 5513 Var. 5598 Var. 5683 Var. 5768 Var. 5853 Var. 5938 Var. 6023 110 Var. 5514 Var. 5599 Var. 5684 Var. 5769 Var. 5854 Var. 5939 Var. 6024 120 Var. 5515 Var. 5600 Var. 5685 Var. 5770 Var. 5855 Var. 5940 Var. 6025 125 Var. 5516 Var. 5601 Var. 5686 Var. 5771 Var. 5856 Var. 5941 Var. 6026 130 Var. 5517 Var. 5602 Var. 5687 Var. 5772 Var. 5857 Var. 5942 Var. 6027 140 Var. 5518 Var. 5603 Var. 5688 Var. 5773 Var. 5858 Var. 5943 Var. 6028 150 Var. 5519 Var. 5604 Var. 5689 Var. 5774 Var. 5859 Var. 5944 Var. 6029 160 Var. 5520 Var. 5605 Var. 5690 Var. 5775 Var. 5860 Var. 5945 Var. 6030 170 Var. 5521 Var. 5606 Var. 5691 Var. 5776 Var. 5861 Var. 5946 Var. 6031 175 Var. 5522 Var. 5607 Var. 5692 Var. 5777 Var. 5862 Var. 5947 Var. 6032 180 Var. 5523 Var. 5608 Var. 5693 Var. 5778 Var. 5863 Var. 5948 Var. 6033 190 Var. 5524 Var. 5609 Var. 5694 Var. 5779 Var. 5864 Var. 5949 Var. 6034 200 Var. 5525 Var. 5610 Var. 5695 Var. 5780 Var. 5865 Var. 5950 Var. 6035 Var. = Variation

1. Sugars and Sugar Alcohols

Advantageously, it was found that the addition of sugar and/or sugar alcohol to furin compositions (e.g., rfurin compositions) increases the storage stability of the composition. For example, as shown in FIGS. 17 to 20 and 22 to 25, the inclusion of as little as 2% sugar and/or sugar alcohol can increase the storage stability of a furin composition by at least 20%. And the inclusion of 10% sugar and/or sugar alcohol can increase the storage stability of a furin composition by at least 75%. Accordingly, in one aspect of the present disclosure, a storage stable composition of furin (e.g., rfurin) contains a stabilizing amount of sugar and/or sugar alcohol.

In one embodiment, the storage stable composition of furin (e.g., rfurin) contains monosaccharide sugar. In a particular embodiment, the monosaccharide sugar is selected from the group consisting of a diose sugar, a triose sugar, a tetrose sugar, a pentose sugar, a hexose sugar, a heptose sugar, and an octose sugar. In a particular embodiment, the sugar is a pentose sugar, a hexose sugar, or a combination thereof. In a specific embodiment, the sugar is a hexose sugar.

In another embodiment, the storage stable composition of furin (e.g., rfurin) contains a disaccharide sugar. In a particular embodiment, the disaccharide sugar is selected from disaccharide sugars formed from pentose and/or hexose monosaccharides. In another particular embodiment, the sugar is selected from disaccharide sugars formed from hexose monosaccharides. In one embodiment, the sugar is sucrose, trehalose, or a combination thereof. In one specific embodiment, the sugar is sucrose. In another specific embodiment, the sugar is trehalose. In one embodiment, the sugar is formulated as trehalose dihydrate.

In another embodiment, the storage stable composition of furin (e.g., rfurin) contains a sugar alcohol. In a particular embodiment, the sugar alcohol is selected from glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In another particular embodiment, the sugar alcohol is mannitol.

In yet another embodiment, the stable composition of furin (e.g., rfurin) contains a mixture of sugar and sugar alcohol. In one embodiment, the mixture contains at least two of a monosaccharide, a disaccharide, and a sugar alcohol. In another embodiment, the mixture contains at least two of a pentose sugar, a hexose sugar, a disaccharide formed from pentose and/or hexose monosaccharides, and a sugar alcohol. In another embodiment, the mixture contains at least two of sucrose, trehalose, and mannitol.

In one embodiment, the sugar or sugar alcohol is present at a concentration of from 2% to 20%, 2% to 17.5%, 2% to 15%, 2% to 12.5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 5% to 20%, 5% to 17.5%, 5% to 15%, 5% to 12.5%, 5% to 10%, 7.5% to 20%, 7.5% to 17.5%, 7.5% to 15%, 7.5% to 12.5%, 10% to 20%, 10% to 17.5%, 10% to 15%, 4±2%, 5±2%, 6±2%, 7±2%, 8±2%, 9±2%, 10±2%, 11±2%, 12±2%, 13±2%, 14±2%, 15±2%, 16±2%, 17±2%, 18±2%, 3±1%, 4±1%, 5±1%, 6±1%, 7±1%, 8±1%, 9±1%, 10±1%, 11±1%, 12±1%, 13±1%, 14±1%, 15±1%, 16±1%, 17±1%, 18±1%, 19±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% trehalose, from 10 ppm to 100 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% trehalose, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10±5% sugar or sugar alcohol, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10±5% sugar or sugar alcohol, from 10 to 100 ppm non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10±5% trehalose, from 10 to 100 ppm non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% trehalose. In another specific embodiment, the composition contains 10±1% trehalose. In another specific embodiment, the composition contains 10% trehalose.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10±5% sugar or sugar alcohol, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the storage stable furin composition further comprises from 10 to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10±5% sugar or sugar alcohol, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH from of 6.0±0.2. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10±5% trehalose, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% trehalose. In another specific embodiment, the composition contains 10±1% trehalose. In another specific embodiment, the composition contains 10% trehalose.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In some embodiments, stabilizing agents used in the formulations of the present disclosure are selected from a group that includes, without limitation: sucrose, trehalose, mannitol, raffinose, and arginine. These agents are present in the formulations of the present invention in an amount of from 0.1% to 20%. In certain embodiments, the stabilizing agent is present in an amount of from 5% to 15%, or about 10%. In further embodiments, formulations of the present disclosure include stabilizing agents in an amount of from 0.5% to 19%, 1% to 18%, 1.5% to 17%, 2.0% to 16%, 2.5% to 15%, 3.0% to 14%, 3.5% to 13%, 4.0% to 12%, 4.5% to 11%, 5.0% to 10%, 5.5% to 9%, or 6.0% to 8%. Certain formulations include mannitol, sucrose, and/or trehalose in combination with one or more of any other formulation components disclosed herein.

2. Non-Ionic Surfactants

Advantageously, it was found that the addition of sugar non-ionic surfactant to furin compositions (e.g., rfurin compositions) increases the stability of the composition to mechanical stress. For example, as shown in FIGS. 31 to 42, the inclusion of as little as 10 ppm non-ionic surfactant can increase the stability of a furin composition subjected to mechanical stress by at least 25%. And the inclusion of 50 ppm non-ionic surfactant can increase the stability of a furin composition subjected to mechanical stress by at least 40%. Accordingly, in one aspect of the present disclosure, a storage stable composition of furin (e.g., rfurin) contains a stabilizing amount of non-ionic surfactant.

In one embodiment, storage stable compositions of furin (e.g., rfurin) are provided which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

In one embodiment, the non-ionic surfactant is present at a concentration of from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 175 ppm, 50±25 ppm, 60±25 ppm, 70±25 ppm, 75±25 ppm, 80±25 ppm, 90±25 ppm, 100±25 ppm, 125±25 ppm, 150±25 ppm, 175±25 ppm, 30±10 ppm, 40±10 ppm, 50±10 ppm, 60±10 ppm, 70±10 ppm, 75±10 ppm, 80±10 ppm, 90±10 ppm, 100±10 ppm, 110±10 ppm, 120±10 ppm, 125±10 ppm, 130±10 ppm, 140±10 ppm, 150±10 ppm, 160±10 ppm, 170±10 ppm, 175±10 ppm, 180±10 ppm, 190±10 ppm, 25 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 75 ppm, 80 ppm, 90 ppm, 100 ppm, 110 ppm, 120 ppm, 125 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 175 ppm, 180 ppm, 190 ppm, or 200 ppm. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic water soluble sorbitan fatty acid ester, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and a non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 10 ppm to 200 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic water soluble sorbitan fatty acid ester, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 75±25% non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, 75±25 ppm non-ionic water soluble sorbitan fatty acid ester, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75±5 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75 ppm non-ionic water soluble sorbitan fatty acid ester.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 75±25% non-ionic surfactant, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the storage stable furin composition further comprises from 2% to 10% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, 91 mM buffering agent, and a pH from of 6.0±0.2. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10% sugar or sugar alcohol, 75±25 ppm non-ionic water soluble sorbitan fatty acid ester, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75±5 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75 ppm non-ionic water soluble sorbitan fatty acid ester.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is 6M. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

The furin (e.g., rfurin) compositions of the present invention also preferably include a surfactant, preferably a non-ionic surfactant, and preferably in an amount from 0.1 ppm to 150 ppm, or from 65 ppm to 80 ppm, or at about 75 ppm. In further embodiments, the furin (e.g., rfurin) compositions include a surfactant in an amount of from 0.5 ppm to 140 ppm, 1.0 ppm to 130 ppm, 10 ppm to 120 ppm, 20 ppm to 110 ppm, 30 ppm to 100 ppm, 40 ppm to 95 ppm, 50 ppm to 90 ppm, 55 ppm to 85 ppm, 60 ppm to 80 ppm, or 70 ppm to 75 ppm. In certain embodiments, the surfactant is chosen from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, pluronic polyols, glycerol, glucamides (such as Mega 8), tritons, and Brij 35 (polyoxyethylene 23 lauryl ether). Several grades of pluronic polyols (sold under the trade name Pluronic, manufactured by the BASF Wyandotte Corporation) are available. These polyols, of diversified molecular weight (from 1,000 to over 16,000) and physicochemical properties have been used as surfactants. Pluronic F-38, of a molecular weight of 5,000 and Pluronic F-68, molecular weight 9,000, both contain (by weight) 80 percent hydrophilic polyoxyethylene groups and 20 percent hydrophobic polyoxypropylene groups. In one embodiment, the surfactant is polysorbate 80. In a particular embodiment, the polysorbate 80 is vegetable-derived polysorbate 80.

B. Pharmaceutically Acceptable Salts

The stabilized compositions of furin (e.g., rfurin) provided herein generally include a pharmaceutically acceptable salt at a concentration tolerated by the furin polypeptide during storage. In one embodiment, the pharmaceutically acceptable salt is a chloride salt. In a specific embodiment, the pharmaceutically acceptable salt is a monovalent chloride salt. In a more specific embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.

In one embodiment, the concentration of pharmaceutically acceptable salt in a stabilized furin (e.g., rfurin) composition provided herein is from 10 mM to 500 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 100 mM to 300 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 150 mM to 250 mM. In yet other embodiments, the concentration of pharmaceutically acceptable salt in the stabilized furin (e.g., rfurin) compositions provided herein is selected from variations 6036 to 6180 found in Table 10.

TABLE 10 Exemplary pharmaceutically acceptable salt concentrations useful for the stabilization of furin (e.g., rfurin) compositions.  10 mM to 500 mM Var. 6036 100 mM to 300 mM Var. 6037 150 mM to 250 mM Var. 6038  50 mM to 500 mM Var. 6039  50 mM to 450 mM Var. 6040  50 mM to 400 mM Var. 6041 100 to 400 mM Var. 6042 100 mM to 350 mM Var. 6043 100 mM to 250 mM Var. 6044 100 mM to 200 mM Var. 6045 150 mM to 400 mM Var. 6046 150 mM to 350 mM Var. 6047 150 mM to 300 mM Var. 6048 150 mM to 250 mM Var. 6049 150 mM to 200 mM Var. 6050 175 mM to 225 mM Var. 6051 175 mM to 200 mM Var. 6052 100 ± 25 mM Var. 6053 150 ± 25 mM Var. 6054 200 ± 25 mM Var. 6055 250 ± 25 mM Var. 6056 300 ± 25 mM Var. 6057 350 ± 25 mM Var. 6058 400 ± 25 mM Var. 6059 450 ± 25 mM Var. 6060 190 ± 150 mM Var. 6061 190 ± 125 mM Var. 6062 190 ± 100 mM Var. 6063 190 ± 90 mM Var. 6064 190 ± 80 mM Var. 6065 190 ± 70 mM Var. 6066 190 ± 60 mM Var. 6067 190 ± 50 mM Var. 6068 190 ± 40 mM Var. 6069 190 ± 30 mM Var. 6070 190 ± 25 mM Var. 6071 190 ± 20 mM Var. 6072 190 ± 15 mM Var. 6073 190 ± 10 mM Var. 6074 190 ± 5 mM Var. 6075 190 ± 2 mM Var. 6076 100 ± 5 mM Var. 6077 105 ± 5 mM Var. 6078 110 ± 5 mM Var. 6079 115 ± 5 mM Var. 6080 120 ± 5 mM Var. 6081 125 ± 5 mM Var. 6082 130 ± 5 mM Var. 6083 135 ± 5 mM Var. 6084 140 ± 5 mM Var. 6085 145 ± 5 mM Var. 6086 150 ± 5 mM Var. 6087 155 ± 5 mM Var. 6088 160 ± 5 mM Var. 6089 165 ± 5 mM Var. 6090 170 ± 5 mM Var. 6091 175 ± 5 mM Var. 6092 180 ± 5 mM Var. 6093 185 ± 5 mM Var. 6094 186 ± 5 mM Var. 6095 187 ± 5 mM Var. 6096 188 ± 5 mM Var. 6097 189 ± 5 mM Var. 6098 190 ± 5 mM Var. 6099 191 ± 5 mM Var. 6100 192 ± 5 mM Var. 6101 193 ± 5 mM Var. 6102 194 ± 5 mM Var. 6103 195 ± 5 mM Var. 6104 196 ± 5 mM Var. 6105 197 ± 5 mM Var. 6106 198 ± 5 mM Var. 6107 199 ± 5 mM Var. 6108 200 ± 5 mM Var. 6109 205 ± 5 mM Var. 6110 210 ± 5 mM Var. 6111 215 ± 5 mM Var. 6112 220 ± 5 mM Var. 6113 225 ± 5 mM Var. 6114 230 ± 5 mM Var. 6115 235 ± 5 mM Var. 6116 240 ± 5 mM Var. 6117 245 ± 5 mM Var. 6118 250 ± 5 mM Var. 6119 255 ± 5 mM Var. 6120 260 ± 5 mM Var. 6121 265 ± 5 mM Var. 6122 270 ± 5 mM Var. 6123 275 ± 5 mM Var. 6124 280 ± 5 mM Var. 6125 285 ± 5 mM Var. 6126 290 ± 5 mM Var. 6127 295 ± 5 mM Var. 6128 300 ± 5 mM Var. 6129 175 mM Var. 6130 176 mM Var. 6131 177 mM Var. 6132 178 mM Var. 6133 179 mM Var. 6134 180 mM Var. 6135 181 mM Var. 6136 182 mM Var. 6137 183 mM Var. 6138 184 mM Var. 6139 185 mM Var. 6140 186 mM Var. 6141 187 mM Var. 6142 188 mM Var. 6143 189 mM Var. 6144 190 mM Var. 6145 191 mM Var. 6146 192 mM Var. 6147 193 mM Var. 6148 194 mM Var. 6149 195 mM Var. 6150 196 mM Var. 6151 197 mM Var. 6152 198 mM Var. 6153 199 mM Var. 6154 200 mM Var. 6155 201 mM Var. 6156 202 mM Var. 6157 203 mM Var. 6158 204 mM Var. 6159 205 mM Var. 6160 206 mM Var. 6161 207 mM Var. 6162 208 mM Var. 6163 209 mM Var. 6164 210 mM Var. 6165 211 mM Var. 6166 212 mM Var. 6167 213 mM Var. 6168 214 mM Var. 6169 215 mM Var. 6170 216 mM Var. 6171 217 mM Var. 6172 218 mM Var. 6173 219 mM Var. 6174 220 mM Var. 6175 221 mM Var. 6176 222 mM Var. 6177 223 mM Var. 6178 224 mM Var. 6179 225 mM Var. 6180 Var. = Variation

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the concentration of the buffering agent is 90±25 mM. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, from 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In some embodiments, monovalent chloride salts, such as sodium chloride and potassium chloride, are used in formulations of the present disclosure. Although the formulation component comprising monovalent chloride salts is described primarily in terms of sodium chloride, it will be appreciated that any monovalent chloride salt, including potassium chloride, may be used in accordance with the descriptions herein for sodium chloride. In certain embodiments, sodium chloride is included in the present formulations at an amount of from 50 to 500 mM. In other embodiments, sodium chloride is included in the formulations at an amount of from 100 to 300 mM, from 150 to 250 mM, or at about 190 mM. In further embodiments, sodium chloride is included in the present formulations in an amount of from 100 to 300 mM, 110 to 280 mM, 120 to 260 mM, 130 to 240 mM, 140 to 220 mM, 150 to 200 mM, or 160 to 180 mM. In a specific embodiment, the formulation includes 190 mM sodium chloride in combination with one or more of any other formulation components disclosed herein.

C. Buffering Agents

Advantageously, it was found that furin (e.g., rfurin) compositions are stabilized at pH 6.0 to 7.0. For example, it is shown in FIGS. 11 and 12 that furin compositions formulated at pH 5.5 are stabilized as compared to furin compositions formulated at pH 5.0. Also shown is that furin compositions formulated at pH 6.0 are further stabilized as compared to compositions formulated at pH 5.5. Furthermore, as shown in FIG. 17, furin (e.g., rfurin) compositions formulated at pH 7.0 have similar stability to compositions formulated at pH 6.0.

Accordingly, in certain embodiments, the stabilized furin (e.g., rfurin) compositions provided herein are formulated at a pH from 5.5 to 7.5. In a specific embodiment, a stabilized furin (e.g., rfurin) composition is formulated at a pH from 6.0 to 7.0. In another specific embodiment, a stabilized furin composition is formulated at pH 6.0±0.2. In a more specific embodiment, a stabilized furin composition is formulated at pH 6.0. In yet other embodiments, a stabilized furin composition is formulated at pH selected from variations 6181 to 6403 found in Table 11.

TABLE 11 Exemplary pH ranges useful for the stabilization of furin (e.g., rfurin) compositions. 5.5-8.0 Var. 6181 5.5-7.9 Var. 6182 5.5-7.8 Var. 6183 5.5-7.6 Var. 6184 5.5-7.5 Var. 6185 5.5-7.4 Var. 6186 5.5-7.3 Var. 6187 5.5-7.2 Var. 6188 5.5-7.1 Var. 6189 5.5-7.0 Var. 6190 5.5-6.9 Var. 6191 5.5-6.8 Var. 6192 5.5-6.7 Var. 6193 5.5-6.6 Var. 6194 5.5-6.5 Var. 6195 5.5-6.4 Var. 6196 5.5-6.3 Var. 6197 5.5-6.2 Var. 6198 5.5-6.1 Var. 6199 5.5-6.0 Var. 6200 5.6-8.0 Var. 6201 5.6-7.9 Var. 6202 5.6-7.8 Var. 6203 5.6-7.6 Var. 6204 5.6-7.5 Var. 6205 5.6-7.4 Var. 6206 5.6-7.3 Var. 6207 5.6-7.2 Var. 6208 5.6-7.1 Var. 6209 5.6-7.0 Var. 6210 5.6-6.9 Var. 6211 5.6-6.8 Var. 6212 5.6-6.7 Var. 6213 5.6-6.6 Var. 6214 5.6-6.5 Var. 6215 5.6-6.4 Var. 6216 5.6-6.3 Var. 6217 5.6-6.2 Var. 6218 5.6-6.1 Var. 6219 5.6-6.0 Var. 6220 5.7-8.0 Var. 6221 5.7-7.9 Var. 6222 5.7-7.8 Var. 6223 5.7-7.6 Var. 6224 5.7-7.5 Var. 6225 5.7-7.4 Var. 6226 5.7-7.3 Var. 6227 5.7-7.2 Var. 6228 5.7-7.1 Var. 6229 5.7-7.0 Var. 6230 5.7-6.9 Var. 6231 5.7-6.8 Var. 6232 5.7-6.7 Var. 6233 5.7-6.6 Var. 6234 5.7-6.5 Var. 6235 5.7-6.4 Var. 6236 5.7-6.3 Var. 6237 5.7-6.2 Var. 6238 5.7-6.1 Var. 6239 5.7-6.0 Var. 6240 5.8-8.0 Var. 6241 5.8-7.9 Var. 6242 5.8-7.8 Var. 6243 5.8-7.6 Var. 6244 5.8-7.5 Var. 6245 5.8-7.4 Var. 6246 5.8-7.3 Var. 6247 5.8-7.2 Var. 6248 5.8-7.1 Var. 6249 5.8-7.0 Var. 6250 5.8-6.9 Var. 6251 5.8-6.8 Var. 6252 5.8-6.7 Var. 6253 5.8-6.6 Var. 6254 5.8-6.5 Var. 6255 5.8-6.4 Var. 6256 5.8-6.3 Var. 6257 5.8-6.2 Var. 6258 5.8-6.1 Var. 6259 5.8-6.0 Var. 6260 5.9-8.0 Var. 6261 5.9-7.9 Var. 6262 5.9-7.8 Var. 6263 5.9-7.6 Var. 6264 5.9-7.5 Var. 6265 5.9-7.4 Var. 6266 5.9-7.3 Var. 6267 5.9-7.2 Var. 6268 5.9-7.1 Var. 6269 5.9-7.0 Var. 6270 5.9-6.9 Var. 6271 5.9-6.8 Var. 6272 5.9-6.7 Var. 6273 5.9-6.6 Var. 6274 5.9-6.5 Var. 6275 5.9-6.4 Var. 6276 5.9-6.3 Var. 6277 5.9-6.2 Var. 6278 5.9-6.1 Var. 6279 5.9-6.0 Var. 6280 6.0-8.0 Var. 6281 6.0-7.9 Var. 6282 6.0-7.8 Var. 6283 6.0-7.6 Var. 6284 6.0-7.5 Var. 6285 6.0-7.4 Var. 6286 6.0-7.3 Var. 6287 6.0-7.2 Var. 6288 6.0-7.1 Var. 6289 6.0-7.0 Var. 6290 6.0-6.9 Var. 6291 6.0-6.8 Var. 6292 6.0-6.7 Var. 6293 6.0-6.6 Var. 6294 6.0-6.5 Var. 6295 6.0-6.4 Var. 6296 6.0-6.3 Var. 6297 6.0-6.2 Var. 6298 6.0-6.1 Var. 6299 5.7 ± 0.2 Var. 6300 5.8 ± 0.2 Var. 6301 5.9 ± 0.2 Var. 6302 6.0 ± 0.2 Var. 6303 6.1 ± 0.2 Var. 6304 6.2 ± 0.2 Var. 6305 6.3 ± 0.2 Var. 6306 6.4 ± 0.2 Var. 6307 6.5 ± 0.2 Var. 6308 6.6 ± 0.2 Var. 6309 6.7 ± 0.2 Var. 6310 6.8 ± 0.2 Var. 6311 6.9 ± 0.2 Var. 6312 7.0 ± 0.2 Var. 6313 7.1 ± 0.2 Var. 6314 7.2 ± 0.2 Var. 6315 7.3 ± 0.2 Var. 6316 7.4 ± 0.2 Var. 6317 7.5 ± 0.2 Var. 6318 7.6 ± 0.2 Var. 6319 7.7 ± 0.2 Var. 6320 7.8 ± 0.2 Var. 6321 5.6 ± 0.1 Var. 6322 5.7 ± 0.1 Var. 6323 5.8 ± 0.1 Var. 6324 5.9 ± 0.1 Var. 6325 6.0 ± 0.1 Var. 6326 6.1 ± 0.1 Var. 6327 6.2 ± 0.1 Var. 6328 6.3 ± 0.1 Var. 6329 6.4 ± 0.1 Var. 6330 6.5 ± 0.1 Var. 6331 6.6 ± 0.1 Var. 6332 6.7 ± 0.1 Var. 6333 6.8 ± 0.1 Var. 6334 6.9 ± 0.1 Var. 6335 7.0 ± 0.1 Var. 6336 7.1 ± 0.1 Var. 6337 7.2 ± 0.1 Var. 6338 7.3 ± 0.1 Var. 6339 7.4 ± 0.1 Var. 6340 7.5 ± 0.1 Var. 6341 7.6 ± 0.1 Var. 6342 7.7 ± 0.1 Var. 6343 7.8 ± 0.1 Var. 6344 5.5 Var. 6345 5.6 Var. 6346 5.7 Var. 6347 5.8 Var. 6348 5.9 Var. 6349 6 Var. 6350 6.1 Var. 6351 6.2 Var. 6352 6.3 Var. 6353 6.4 Var. 6354 6.5 Var. 6355 6.6 Var. 6356 6.7 Var. 6357 6.8 Var. 6358 6.9 Var. 6359 7 Var. 6360 7.1 Var. 6361 7.2 Var. 6362 7.3 Var. 6363 7.4 Var. 6364 7.5 Var. 6365 7.6 Var. 6366 7.7 Var. 6367 7.8 Var. 6368 7.9 Var. 6369 8 Var. 6370 6.0 ± 0.3 Var. 6371 6.0 ± 0.4 Var. 6372 6.0 ± 0.5 Var. 6373 6.1 ± 0.3 Var. 6374 6.1 ± 0.4 Var. 6375 6.1 ± 0.5 Var. 6376 6.2 ± 0.3 Var. 6377 6.2 ± 0.4 Var. 6378 6.2 ± 0.5 Var. 6379 6.3 ± 0.3 Var. 6380 6.3 ± 0.4 Var. 6381 6.3 ± 0.5 Var. 6382 6.4 ± 0.3 Var. 6383 6.4 ± 0.4 Var. 6384 6.4 ± 0.5 Var. 6385 6.5 ± 0.3 Var. 6386 6.5 ± 0.4 Var. 6387 6.5 ± 0.5 Var. 6388 6.6 ± 0.3 Var. 6389 6.6 ± 0.4 Var. 6390 6.6 ± 0.5 Var. 6391 6.7 ± 0.3 Var. 6392 6.7 ± 0.4 Var. 6393 6.7 ± 0.5 Var. 6394 6.8 ± 0.3 Var. 6395 6.8 ± 0.4 Var. 6396 6.8 ± 0.5 Var. 6397 6.9 ± 0.3 Var. 6398 6.9 ± 0.4 Var. 6399 6.9 ± 0.5 Var. 6400 7.0 ± 0.3 Var. 6401 7.0 ± 0.4 Var. 6402 7.0 ± 0.5 Var. 6403 Var. = Variation

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, from 90±25 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 191 mM of a pharmaceutically acceptable salt, 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, the stabilized furin (e.g., rfurin) compositions provided herein are formulated at an appropriate pH using one or more buffering agents. In one embodiment, the one or more buffering agent is selected from histidine, imidazole, phosphate, citrate, Tris, acetate (e.g., acetic acid), BIS-Tris Propane, PIPES, MOPS, HEPES, MES, ACES, and a combination thereof. In a particular embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES.

Buffering agent is present in the stabilized furin (e.g., rfurin) compositions at a concentration suitable to maintain the pH of the composition during storage over a period of time (e.g., weeks, months, or years). In one embodiment, the concentration of buffering agent in the formulation is from 10 mM to 300 mM. In another embodiment, the concentration of buffering agent in the formulation is between 10 mM to 200 mM. In a specific embodiment, the concentration of buffering agent in the formulation is 90±25 mM. In yet other embodiments, the concentration of buffering agent in the composition is selected from variations 6404 to 6469 found in Table 12.

TABLE 12 Exemplary buffering agent concentrations (mM) useful for the stabilization of furin (e.g., rfurin) compositions. 10-300 Var. 6404 10-275 Var. 6405 10-250 Var. 6406 10-225 Var. 6407 10-200 Var. 6408 10-175 Var. 6409 10-150 Var. 6410 10-125 Var. 6411 10-100 Var. 6412 25-300 Var. 6413 25-275 Var. 6414 25-250 Var. 6415 25-225 Var. 6416 25-200 Var. 6417 25-175 Var. 6418 25-150 Var. 6419 25-125 Var. 6420 25-100 Var. 6421 50-300 Var. 6422 50-275 Var. 6423 50-250 Var. 6424 50-225 Var. 6425 50-200 Var. 6426 50-175 Var. 6427 50-150 Var. 6428 50-125 Var. 6429 50-100 Var. 6430 75-300 Var. 6431 75-275 Var. 6432 75-250 Var. 6433 75-225 Var. 6434 75-200 Var. 6435 75-175 Var. 6436 75-150 Var. 6437 75-125 Var. 6438 75-100 Var. 6439 90 ± 75 Var. 6440 90 ± 50 Var. 6441 90 ± 25 Var. 6442 90 ± 10 Var. 6443 90 ± 5  Var. 6444 90 ± 2  Var. 6445 91 ± 10 Var. 6446 91 ± 5  Var. 6447 91 ± 2  Var. 6448 80 Var. 6449 81 Var. 6450 82 Var. 6451 83 Var. 6452 84 Var. 6453 85 Var. 6454 86 Var. 6455 87 Var. 6456 88 Var. 6457 89 Var. 6458 90 Var. 6459 91 Var. 6460 92 Var. 6461 93 Var. 6462 94 Var. 6463 95 Var. 6464 96 Var. 6465 97 Var. 6466 98 Var. 6467 99 Var. 6468 100  Var. 6469 Var. = Variation

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 191 mM of a pharmaceutically acceptable salt, 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.

Buffers may also be present in formulations of the invention in combination with one or more of any other formulation components described herein. As shown in the Examples, the highly stabilized formulations of the present invention show increased stability at pH 6.0. In certain embodiments, the pH of the highly stabilized formulations should preferably be maintained in the range of from 6.0 to 8.0, or at a pH of about 6.0. The buffering agent can be any physiologically acceptable chemical entity or combination of chemical entities which have the capacity to act as buffers, including, without limitation: histidine, imidazole, phosphate, citrate, Tris, Acetate, BIS-Tris Propane, PIPES, MOPS, HEPES, MES, and ACES. The full chemical designations of many of these buffering agents are listed in Table 1 below. In certain embodiments, if calcium is present in the formulation at a concentration above about 5 mM, phosphate is not used as a buffering agent. In some embodiments, the buffering agent is included in a concentration of from 10 mM to 200 mM, or 10 to 100 mM, or 30-60 mM, or about 46 mM. In further embodiments, an individual buffering agent is included in a concentration of from 15 to 95 mM, 20 to 90 mM, 25 to 85 mM, 30 to 80 mM, 35 to 75 mM, 40 to 70 mM, 45 to 65 mM, or 50 to 60 mM. In certain embodiments, the formulation contains two buffering agents.

TABLE 13 Exemplary Buffering Agents Tris tris-(hydroxymethyl)-aminomethane BIS-Tris Propane 1,3-bis-[tris-(hydroxy-methyl)methylamino]-propane PIPES piperazine-N,N′-bis-(2-ethanesulfonic acid) MOPS 3-{N-morpholino) propanesulfonic acid HEPES N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid MES 2-(N-morpholino) ethanesulfonic acid ACES N-2-acetamido-2-aminoethanesulfonic acid

In certain aspects the present formulations include a carboxylic acid in combination with one or more of the formulation components described herein. In further aspects, the carboxylic acid is preferably acetic acid (e.g., acetate). In certain embodiments, the formulation includes from 20 mM to 100 mM acetic acid, or from 30 mM to 50 mM, or about 45 mM acetic acid or any other carboxylic acid. In further embodiments, the formulation includes from 25 mM to 90 mM, 30 mM to 80 mM, 35 mM to 70 mM, 40 mM to 60 mM, or 45 mM to 50 mM acetic acid or any other carboxylic acid.

D. Additional Formulation Components

The highly stabilized formulations of the present disclosure include furin, e.g., rfurin, and one or more of stabilizing agents, buffering agents, sodium chloride, salts, and other excipients. Such components are described in further detail below. As will be appreciated, any of the formulation components described herein can be used singly or in any combination.

The highly stabilized Furin formulations of the present disclosure show increased stability toward shear (agitation), lyophilization, and freeze/thaw stress as well as resistance to product loss or denaturation at container surfaces as compared to control or starting formulations.

Furin is included in formulations of the present disclosure in concentrations from 5,000 to 500,000 U/mL. In certain embodiments, Furin is included in concentrations from 5,500 to 55,000, from 6,000 to 50,000, from 6,500 to 45,000, from 7,000 to 40,000, from 7,500 to 35,000, from 8,000 to 30,000, from 8,500 to 25,000, from 9,000 to 20,000, from 9,500 to 15,000, and about 10,000 U/mL. In yet other embodiments, furin is included in the formulations of the present disclosure at a concentration selected from variations 6470 to 6533 found in Table 14, reported in thousands of Units of furin activity per mL. In specific embodiments, the furin contained in the highly stabilized formulations of the present disclosure is rfurin.

TABLE 14 Exemplary furin (e.g., rfurin) concentrations (expressed in thousands of Units furin activity per mL) used in the stabilized compositions provided herein.  1-1,000 Var. 6470 1-900 Var. 6471 1-800 Var. 6472 1-700 Var. 6473 1-600 Var. 6474 1-500 Var. 6475 1-450 Var. 6476 1-400 Var. 6477 1-350 Var. 6478 1-300 Var. 6479 1-250 Var. 6480 1-200 Var. 6481 1-150 Var. 6482 1-100 Var. 6483 1-52  Var. 6484 1-50  Var. 6485  5-1,000 Var. 6486 5-900 Var. 6487 5-800 Var. 6488 5-700 Var. 6489 5-600 Var. 6490 5-500 Var. 6491 5-450 Var. 6492 5-400 Var. 6493 5-350 Var. 6494 5-300 Var. 6495 5-250 Var. 6496 5-200 Var. 6497 5-150 Var. 6498 5-100 Var. 6499 5-52  Var. 6500 5-50  Var. 6501  8-1,000 Var. 6502 8-900 Var. 6503 8-800 Var. 6504 8-700 Var. 6505 8-600 Var. 6506 8-500 Var. 6507 8-450 Var. 6508 8-400 Var. 6509 8-350 Var. 6510 8-300 Var. 6511 8-250 Var. 6512 8-200 Var. 6513 8-150 Var. 6514 8-100 Var. 6515 8-52  Var. 6516 8-50  Var. 6517  10-1,000 Var. 6518 10-900  Var. 6519 10-800  Var. 6520 10-700  Var. 6521 10-600  Var. 6522 10-500  Var. 6523 10-450  Var. 6524 10-400  Var. 6525 10-350  Var. 6526 10-300  Var. 6527 10-250  Var. 6528 10-200  Var. 6529 10-150  Var. 6530 10-100  Var. 6531 10-52  Var. 6532 10-50  Var. 6533 Var. = Variation

In another embodiment, the furin (e.g., rfurin) concentration of a stabilized composition provided herein can be expressed as the mass of furin present in the composition. In one embodiment, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 100 ng/mL to 100 mg/mL. In another embodiment, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 1 μg/mL to 10 mg/mL. In another embodiment, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 1 μg/mL to 1 mg/mL. In yet other embodiments, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration selected from variations 6534 to 6638 found in Table 15. In specific embodiments, the furin contained in the highly stabilized formulations of the present disclosure is rfurin.

TABLE 15 Exemplary furin (e.g., rfurin) concentrations used in the stabilized compositions provided herein. 100 ng/mL to 100 mg/mL  Var. 6534 100 ng/mL to 50 mg/mL  Var. 6535 100 ng/mL to 25 mg/mL  Var. 6536 100 ng/mL to 10 mg/mL  Var. 6537 100 ng/mL to 5 mg/mL   Var. 6538 100 ng/mL to 1 mg/mL   Var. 6539 100 ng/mL to 500 μg/mL  Var. 6540 100 ng/mL to 250 μg/mL  Var. 6541 100 ng/mL to 100 μg/mL  Var. 6542 100 ng/mL to 50 μg/mL  Var. 6543 100 ng/mL to 25 μg/mL  Var. 6544 100 ng/mL to 10 μg/mL  Var. 6545 100 ng/mL to 5 μg/mL   Var. 6546 100 ng/mL to 1 μg/mL   Var. 6547  1 μg/mL to 100 mg/mL Var. 6548  1 μg/mL to 50 mg/mL Var. 6549  1 μg/mL to 25 mg/mL Var. 6550  1 μg/mL to 10 mg/mL Var. 6551 1 μg/mL to 5 mg/mL  Var. 6552 1 μg/mL to 1 mg/mL  Var. 6553  1 μg/mL to 500 μg/mL Var. 6554  1 μg/mL to 250 μg/mL Var. 6555  1 μg/mL to 100 μg/mL Var. 6556 1 μg/mL to 50 μg/mL Var. 6557 1 μg/mL to 25 μg/mL Var. 6558 1 μg/mL to 10 μg/mL Var. 6559 1 μg/mL to 5 μg/mL  Var. 6560  5 μg/mL to 100 mg/mL Var. 6561  5 μg/mL to 50 mg/mL Var. 6562  5 μg/mL to 25 mg/mL Var. 6563  5 μg/mL to 10 mg/mL Var. 6564 5 μg/mL to 5 mg/mL  Var. 6565 5 μg/mL to 1 mg/mL  Var. 6566  5 μg/mL to 500 μg/mL Var. 6567  5 μg/mL to 250 μg/mL Var. 6568  5 μg/mL to 100 μg/mL Var. 6569 5 μg/mL to 50 μg/mL Var. 6570 5 μg/mL to 25 μg/mL Var. 6571 5 μg/mL to 10 μg/mL Var. 6572  10 μg/mL to 100 mg/mL Var. 6573 10 μg/mL to 50 mg/mL  Var. 6574 10 μg/mL to 25 mg/mL  Var. 6575 10 μg/mL to 10 mg/mL  Var. 6576 10 μg/mL to 5 mg/mL  Var. 6577 10 μg/mL to 1 mg/mL  Var. 6578 10 μg/mL to 500 μg/mL Var. 6579 10 μg/mL to 250 μg/mL Var. 6580 10 μg/mL to 100 μg/mL Var. 6581 10 μg/mL to 50 μg/mL  Var. 6582 10 μg/mL to 25 μg/mL  Var. 6583  25 μg/mL to 100 mg/mL Var. 6584 25 μg/mL to 50 mg/mL  Var. 6585 25 μg/mL to 25 mg/mL  Var. 6586 25 μg/mL to 10 mg/mL  Var. 6587 25 μg/mL to 5 mg/mL  Var. 6588 25 μg/mL to 1 mg/mL  Var. 6589 25 μg/mL to 500 μg/mL Var. 6590 25 μg/mL to 250 μg/mL Var. 6591 25 μg/mL to 100 μg/mL Var. 6592 25 μg/mL to 50 μg/mL  Var. 6593  50 μg/mL to 100 mg/mL Var. 6594 50 μg/mL to 50 mg/mL  Var. 6595 50 μg/mL to 25 mg/mL  Var. 6596 50 μg/mL to 10 mg/mL  Var. 6597 50 μg/mL to 5 mg/mL  Var. 6598 50 μg/mL to 1 mg/mL  Var. 6599 50 μg/mL to 500 μg/mL Var. 6600 50 μg/mL to 250 μg/mL Var. 6601 50 μg/mL to 100 μg/mL Var. 6602 100 μg/mL to 100 mg/mL  Var. 6603 100 μg/mL to 50 mg/mL  Var. 6604 100 μg/mL to 25 mg/mL  Var. 6605 100 μg/mL to 10 mg/mL  Var. 6606 100 μg/mL to 5 mg/mL   Var. 6607 100 μg/mL to 1 mg/mL   Var. 6608 100 μg/mL to 500 μg/mL  Var. 6609 100 μg/mL to 250 μg/mL  Var. 6610 250 μg/mL to 100 mg/mL  Var. 6611 250 μg/mL to 50 mg/mL  Var. 6612 250 μg/mL to 25 mg/mL  Var. 6613 250 μg/mL to 10 mg/mL  Var. 6614 250 μg/mL to 5 mg/mL   Var. 6615 250 μg/mL to 1 mg/mL   Var. 6616 250 μg/mL to 500 μg/mL  Var. 6617 500 μg/mL to 100 mg/mL  Var. 6618 500 μg/mL to 50 mg/mL  Var. 6619 500 μg/mL to 25 mg/mL  Var. 6620 500 μg/mL to 10 mg/mL  Var. 6621 500 μg/mL to 5 mg/mL   Var. 6622 500 μg/mL to 1 mg/mL   Var. 6623  1 mg/mL to 100 mg/mL Var. 6624 1 mg/mL to 50 mg/mL Var. 6625 1 mg/mL to 25 mg/mL Var. 6626 1 mg/mL to 10 mg/mL Var. 6627 1 mg/mL to 5 mg/mL  Var. 6628  5 mg/mL to 100 mg/mL Var. 6629 5 mg/mL to 50 mg/mL Var. 6630 5 mg/mL to 25 mg/mL Var. 6631 5 mg/mL to 10 mg/mL Var. 6632 10 mg/mL to 100 mg/mL Var. 6633 10 mg/mL to 50 mg/mL  Var. 6634 10 mg/mL to 25 mg/mL  Var. 6635 25 mg/mL to 100 mg/mL Var. 6636 25 mg/mL to 50 mg/mL  Var. 6637 50 mg/mL to 100 mg/mL Var. 6638 Var. = Variation

In some aspects of the present disclosure, furin (e.g., rfurin) formulations include calcium or another divalent metal cations. In further aspects, the divalent cation is present as a salt, preferably a chloride salt. In certain embodiments, from 0.1 mM to 10 mM of a divalent cation salt can be used, or from 0.5 mM to 2 mM, or about 0.92 mM. In still further embodiments, from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1.5 mM to 7 mM, from 2 mM to 6 mM, from 2.5 mM to 5 mM, or from 3 mM to 4.5 mM divalent cation salt is used in formulations of the invention. When calcium salt is used, it is preferably calcium chloride, but can also be other calcium salts such as calcium gluconate, calcium glubionate, or calcium gluceptate. Divalent cations, including calcium, can be included in combination with one or more other formulation components disclosed herein. Non-limiting examples of divalent metal cations useful in the formulations provided herein include calcium, barium, manganese, magnesium, cobalt, copper, nickel, and zinc.

In some embodiments, the furin (e.g., rfurin) formulations include an antioxidant. The addition of antioxidants to aqueous and lyophilized formulations has been found to improve the stability of these formulations, and thus extend their shelf lives. The antioxidants used must be compatible for use with a pharmaceutical preparation, and in addition are preferably water soluble. When adding antioxidants to a formulation, it is preferable to add such antioxidants as late in the process prior to lyophilization as possible, in order to avoid spontaneous oxidation of the antioxidant. Table 2 below lists suitable antioxidants, which are available commercially through companies such as Calbiochem and Sigma.

TABLE 16 Exemplary Antioxidants N-Acetyl-L-Cysteine/Homocysteine Glutathione 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) Lipoic acid Methionine Sodium Thiosulfate Platinum Glycme-glycine-histidme (tripeptide) Butylatedhydroxytoluene (BHT)

Concentrations in the range of about 0.05 mg/ml to more than 1.0 mg/ml of antioxidants can be used, and it is believed that higher concentrations would also be useful (up to the point of any toxic effects or adverse manufacturing effects, such as a depression of the glass transition temperature of the lyophilized product). Accordingly, in one embodiment, the furin (e.g., rfurin) composition comprises from 0.05 mg/mL to 1.0 mg/mL antioxidant. In other embodiments, the furin (e.g., rfurin) composition comprises from 0.05 to 0.5 mg/mL, 0.1 mg/mL to 0.9 mg/mL, 0.1 mg/mL to 0.8 mg/mL, 0.1 mg/mL to 0.7 mg/mL, 0.1 mg/mL to 0.6 mg/mL, 0.1 mg/mL to 0.5 mg/mL, 0.1 mg/mL to 0.4 mg/mL, 0.1 mg/mL to 0.3 mg/mL, 0.1 mg/mL to 0.2 mg/mL, 0.2 mg/mL to 0.9 mg/mL, 0.2 mg/mL to 0.8 mg/mL, 0.2 mg/mL to 0.7 mg/mL, 0.2 mg/mL to 0.6 mg/mL, 0.2 mg/mL to 0.5 mg/mL, 0.2 mg/mL to 0.4 mg/mL, 0.2 mg/mL to 0.3 mg/mL, 0.3 mg/mL to 0.7 mg/mL, 0.4 mg/mL to 0.6 mg/mL antioxidant.

E. Formulation Development

In certain aspects of the present disclosure, highly stabilized formulations of furin (e.g., rfurin) are prepared by providing a starting (control) furin formulation and adding components to achieve a desired level of component concentrations. For example, the addition of one part 750 ppm non-ionic surfactant to 9 parts furin composition lacking non-ionic surfactant to provide a final formulation comprising 75 ppm non-ionic surfactant. This process is also called “spiking in” to a starting formulation.

In certain embodiments, to produce the highly stabilized furin (e.g., rfurin) formulations of the present disclosure, furin is contained in a starting formulation and a buffer composition is spiked into the starting formulation. In other embodiments, the highly stabilized furin (e.g., rfurin) formulation is formed using dialysis according to methods known in the art. In some embodiments, the methods of formulation involve spiking a concentrated buffer composition into a starting formulation of furin. In one embodiment, polysorbate 80 is added to the starting formulation prior to the addition of other components, particularly prior to the addition of trehalose, in order to protect Furin against agitation during mixing.

In an exemplary embodiment, furin (e.g., rfurin) bulk drug substance (BDS) is contained in a starting formulation of 10 mM sodium acetate, 230 mM sodium chloride, 1 mM calcium chloride, pH 6.0. In one embodiment, the following components are spiked into the starting formulation: 1% polysorbate 80, 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl₂, pH 6.0 and trehalose dihydrate powder to attain the following composition: 46 mM HEPES, 45 mM acetic acid, 190 mM NaCl, 0.92 mM CaCl2, 75 ppm polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0. The increase in acetic acid and the addition of HEPES results in greater stability of the pH. Although at pH 6.0 both acetic acid and HEPES are outside of their highest buffering capacity, the high concentration of these chemicals greatly increases the buffering capacity of the highly stabilized formulation. In certain embodiments, the polysorbate 80 is added to the starting formulation prior to the other components to protect furin from aggregation and adsorption to container surfaces during mixing when the other reagents are added. In certain embodiments, the furin in the starting formulation is mixed with polysorbate 80 and/or any other components except for trehalose. Trehalose and any remaining components are added after polysorbate 80 is added to protect furin from aggregation and adsorption during mixing.

In certain embodiments, the stabilized formulation is combined with a diluent so that the formulation can be used in methods such as an rVWF maturation process. In further embodiments, a stabilized diluent is prepared by spiking a starting diluent (50 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, pH 7.0) with polysorbate 80 to 75 ppm. The combination of the highly stabilized furin (e.g., rfurin) formulation of the present disclosure with this stabilized diluent increases furin recovery in the rVWF maturation step by three to four times compared to the use of the starting furin and diluent formulations.

In certain embodiments, the highly stabilized furin formulations of the present disclosure are lyophilized. During lyophilization, the formulation is converted from being in an aqueous phase to being in an amorphous solid phase, which is thought to protect the protein from chemical and/or conformational instability. In further embodiments, the lyophilized preparation not only contains an amorphous phase, but also includes a component which crystallizes during lyophilization.

One or more components of highly stabilized formulations may in some embodiments be dispersed in the amorphous phase of the lyophilized cake. In addition, the apparent glass transition temperature (Tg′) of the amorphous phase is preferably relatively high during freeze-drying, and the glass transition temperature (Tg) of the solid is likewise preferably high during storage.

IV. Methods for Diluting Aqueous Compositions of Recombinant Furin

In one aspect, the present disclosure provides methods for diluting aqueous compositions of furin (e.g., rfurin). The following embodiments are based in part on the discovery that inclusion of a non-ionic surfactant in a buffer used to dilute furin (e.g., rfurin) results in the recovery of 3-4 times more furin activity as compared to dilution with a buffer lacking the non-ionic surfactant.

In one embodiment, the method includes adding a dilution buffer containing non-ionic surfactant to a furin (e.g., rfurin) composition, to form a diluted furin (e.g., rfurin) composition. In some embodiments, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 1,000:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 500:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 250:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 200:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 100:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 50:1 (dilution buffer:furin composition).

In one embodiment, the method comprises a first step of adding a non-ionic surfactant to a furin (e.g., rfurin) composition and a second step of adding a dilution buffer to the furin (e.g., rfurin) composition containing the non-ionic surfactant, to form a diluted furin composition. In one embodiment, the surfactant will be added to the furin composition at a final concentration of X-fold a desired concentration in the diluted furin composition, where X is the dilution factor. For example, if a final concentration of 10 ppm non-ionic surfactant is desired in a furin composition to be diluted 100-fold, the non-ionic surfactant is added to the starting furin composition at a final concentration of 1,000 ppm (10 ppm×100-fold dilution), and the composition is subsequently diluted by adding 99 parts dilution buffer per 1 part starting solution (accounting for volume added during addition of the non-ionic surfactant).

In certain embodiments, the furin (e.g., rfurin) composition is diluted from 1-fold to 1,000-fold, from 1-fold to 500-fold, from 1-fold to 250-fold, from 1-fold to 200-fold, from 1-fold to 100-fold, from 1-fold to 50-fold, from 1-fold to 10-fold, from 10-fold to 1,000-fold, from 10-fold to 500-fold, from 10-fold to 250-fold, from 10-fold to 200-fold, from 10-fold to 100-fold, from 10-fold to 50-fold, from 50-fold to 1,000-fold, from 50-fold to 500-fold, from 50-fold to 250-fold, from 50-fold to 200-fold, from 50-fold to 100-fold, from 100-fold to 1,000-fold, from 100-fold to 500-fold, from 100-fold to 250-fold, from 100-fold to 200-fold, from 200-fold to 1,000-fold, from 200-fold to 500-fold, or from 200-fold to 250-fold.

A. Furin Dilution Buffer

In one embodiment, the furin dilution buffer will include a pharmaceutically acceptable salt, non-ionic surfactant, and a buffering agent. In one embodiment, the dilution buffer further includes calcium. In another embodiment, the dilution buffer further includes a sugar and/or sugar alcohol.

In one embodiment, pharmaceutically acceptable salt is present in the dilution buffer at a concentration of from 10 mM to 500 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 100 mM to 300 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 150 mM to 250 mM. In yet other embodiments, the concentration of pharmaceutically acceptable salt in the furin dilution buffer is selected from variations 6036 to 6180 found in Table 10. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride.

In one embodiment, the dilution buffer has a pH of from 5.5 to 7.5. In one embodiment, the pH of the dilution buffer is from 5.5 to 7.0. In another embodiment, the pH of the dilution buffer is from 5.5 to 6.5. In another embodiment, the pH of the dilution buffer is 6.0±0.2. In a specific embodiment, the pH of the dilution buffer is 6.0. In other embodiments, the dilution buffer has a pH selected from variations 6181 to 6403 found in Table 11.

In one embodiment, buffering agent is present in the dilution buffer at a concentration of from 10 mM to 300 mM. In another embodiment, the concentration of buffering agent in the dilution buffer is between 10 mM to 200 mM. In other embodiments, the concentration of buffering agent in the dilution buffer is selected from variations 6404 to 6469 found in Table 12. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES.

In some embodiments, the furin dilution buffer includes calcium or another divalent metal cation. In one embodiment, the divalent cation is added as a salt, preferably a chloride salt. In certain embodiments, the concentration of divalent cation (e.g., calcium) in the dilution buffer is from 0.1 mM to 10 mM, from 0.5 mM to 2 mM, or about 0.92 mM. In still further embodiments, from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1.5 mM to 7 mM, from 2 mM to 6 mM, from 2.5 mM to 5 mM, or from 3 mM to 4.5 mM divalent cation is used in the dilution buffer. When calcium salt is used, it is preferably calcium chloride, but can also be other calcium salts such as calcium gluconate, calcium glubionate, or calcium gluceptate. Divalent cations, including calcium, can be included in combination with one or more other formulation components disclosed herein. Non-limiting examples of divalent metal cations useful in the dilution buffers provided herein include calcium, barium, manganese, magnesium, cobalt, copper, nickel, and zinc.

In some embodiments, the furin dilution buffer includes a sugar and/or sugar alcohol. In one embodiment, the dilution buffer contains a monosaccharide sugar. In a particular embodiment, the monosaccharide sugar is selected from the group consisting of a diose sugar, a triose sugar, a tetrose sugar, a pentose sugar, a hexose sugar, a heptose sugar, and an octose sugar. In a particular embodiment, the sugar is a pentose sugar, a hexose sugar, or a combination thereof. In a specific embodiment, the sugar is a hexose sugar.

In another embodiment, the dilution buffer contains a disaccharide sugar. In a particular embodiment, the disaccharide sugar is selected from disaccharide sugars formed from pentose and/or hexose monosaccharides. In another particular embodiment, the sugar is selected from disaccharide sugars formed from hexose monosaccharides. In one embodiment, the sugar is sucrose, trehalose, or a combination thereof. In one specific embodiment, the sugar is sucrose. In another specific embodiment, the sugar is trehalose. In one embodiment, the sugar is formulated as trehalose dihydrate.

In another embodiment, the dilution buffer contains a sugar alcohol. In a particular embodiment, the sugar alcohol is selected from glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In another particular embodiment, the sugar alcohol is mannitol.

In yet another embodiment, the dilution buffer contains a mixture of sugar and sugar alcohol. In one embodiment, the mixture contains at least two of a monosaccharide, a disaccharide, and a sugar alcohol. In another embodiment, the mixture contains at least two of a pentose sugar, a hexose sugar, a disaccharide formed from pentose and/or hexose monosaccharides, and a sugar alcohol. In another embodiment, the mixture contains at least two of sucrose, trehalose, and mannitol.

In one embodiment, the sugar or sugar alcohol is present in the dilution buffer at a concentration of from 2% to 20%, 2% to 17.5%, 2% to 15%, 2% to 12.5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 5% to 20%, 5% to 17.5%, 5% to 15%, 5% to 12.5%, 5% to 10%, 7.5% to 20%, 7.5% to 17.5%, 7.5% to 15%, 7.5% to 12.5%, 10% to 20%, 10% to 17.5%, 10% to 15%, 4±2%, 5±2%, 6±2%, 7±2%, 8±2%, 9±2%, 10±2%, 11±2%, 12±2%, 13±2%, 14±2%, 15±2%, 16±2%, 17±2%, 18±2%, 3±1%, 4±1%, 5±1%, 6±1%, 7±1%, 8±1%, 9±1%, 10±1%, 11±1%, 12±1%, 13±1%, 14±1%, 15±1%, 16±1%, 17±1%, 18±1%, 19±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose.

In one embodiment, storage stable compositions of furin (e.g., rfurin) are provided which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

1. Non-Ionic Surfactants

In one embodiment, the furin dilution buffer, or non-ionic surfactant solution added prior to dilution, contains a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

In one embodiment, the non-ionic surfactant is present in the dilution buffer at a concentration of from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 175 ppm, 50±25 ppm, 60±25 ppm, 70±25 ppm, 75±25 ppm, 80±25 ppm, 90±25 ppm, 100±25 ppm, 125±25 ppm, 150±25 ppm, 175±25 ppm, 30±10 ppm, 40±10 ppm, 50±10 ppm, 60±10 ppm, 70±10 ppm, 75±10 ppm, 80±10 ppm, 90±10 ppm, 100±10 ppm, 110±10 ppm, 120±10 ppm, 125±10 ppm, 130±10 ppm, 140±10 ppm, 150±10 ppm, 160±10 ppm, 170±10 ppm, 175±10 ppm, 180±10 ppm, 190±10 ppm, 25 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 75 ppm, 80 ppm, 90 ppm, 100 ppm, 110 ppm, 120 ppm, 125 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 175 ppm, 180 ppm, 190 ppm, or 200 ppm. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

In one embodiment, the target concentration of non-ionic surfactant after dilution is from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 175 ppm, 50±25 ppm, 60±25 ppm, 70±25 ppm, 75±25 ppm, 80±25 ppm, 90±25 ppm, 100±25 ppm, 125±25 ppm, 150±25 ppm, 175±25 ppm, 30±10 ppm, 40±10 ppm, 50±10 ppm, 60±10 ppm, 70±10 ppm, 75±10 ppm, 80±10 ppm, 90±10 ppm, 100±10 ppm, 110±10 ppm, 120±10 ppm, 125±10 ppm, 130±10 ppm, 140±10 ppm, 150±10 ppm, 160±10 ppm, 170±10 ppm, 175±10 ppm, 180±10 ppm, 190±10 ppm, 25 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 75 ppm, 80 ppm, 90 ppm, 100 ppm, 110 ppm, 120 ppm, 125 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 175 ppm, 180 ppm, 190 ppm, or 200 ppm. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

In one embodiment, the inclusion of a non-ionic surfactant in a furin dilution buffer, or addition prior to dilution, increases the furin activity recovered in a diluted furin composition by at least 10%, as compared to the activity recovered in a furin composition diluted without use of the non-ionic surfactant, or with the use of the non-ionic surfactant at a lower concentration. In other embodiments, the furin activity recovered after dilution using a non-ionic surfactant is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 2-fold, 3-fold, 4-fold, 5-fold, or more greater than the furin activity recovered after dilution without the non-ionic surfactant, or using a lower concentration of the non-ionic surfactant.

V. Stability Assays

As discussed herein, the highly stabilized furin (e.g., rfurin) formulations of the present disclosure show improved stability as compared to control formulations. In one embodiment, improved stability includes retention of a higher percentage of activity than control formulations in various stability assays. Such assays can be used to determine if a formulation is a highly stabilized formulation. In some embodiments, the highly stabilized formulation has at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater activity than a control formulation when assessed by any of the stability assays discussed herein or known in the art.

In further aspects, furin (e.g., rfurin) formulations are tested under stressor conditions, such as storage at high temperature, agitation, freeze/thaw cycles, or some combination thereof. After such stressors, the formulations are assayed using any of the methods described herein or known in the art to determine the stability under these conditions.

In one aspect, furin activity assays are used to assess the stability of a formulation. In some embodiments, a furin activity assays involve a measurement of substrate cleavage. In one embodiment, the substrate is Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; AMC=7-amino-4-methoxy coumarin), which is cleaved by furin, liberating 7-amino-4-methoxy coumarin. The assay is performed, for example, on an ELISA plate. The furin activity in a sample is determined from a standard curve created, for example, from a quality control (QC) furin standard. The activity values are generally calculated as mean values from two dilutions, each in duplicate.

In another aspect, size exclusion chromatography (SEC) is used to evaluate the stability of a furin (e.g., rfurin) formulation. In such assays, stability is indicated by the height of the peak corresponding to monomeric furin. A reduction in monomer peak height means the product is being lost (unstable) and the generation of higher molecular weight peaks indicates that aggregation is occurring and lower molecular weight peaks means the product is degrading. In an exemplary embodiment, after 4 days at 37° C., the relative peak height of a control furin formulation was 58.5%, but in a highly stabilized formulation, the relative peak height was 74.8% (FIG. 48). Thus, the highly stabilized formulation maintained the stability of furin in the composition and retained a larger percentage of the peak height as compared to that seen prior to storage at four days at a higher (37° C.) temperature.

In another aspect, qualitative assays such as Western Blot analyses, are used to evaluate the stability of a Furin formulation. For example, FIG. 26 shows that after 5 days at 35° C., a greater percent of the signal is present as the intact furin molecule rather than the degraded species in the stabilized formulation (lane 11), as compared to a non-stabilized formulation (lane 5).

In still another aspect, qualitative assays such as UV spectra are used to evaluate stability. Such assays are particularly useful when the stressor used is agitation. The UV spectra can indicate the presence of aggregate—the presence of an aggregate is an indication of instability. For example, FIG. 36 shows a major displacement of the spectra upward (indicating aggregation) when the control formulation is agitated. FIGS. 41 and 42 show the protective power of a highly stabilized formulation in that, with agitation, the spectra of the agitated sample is not displaced upward, but overlays the non-agitated sample, which indicates that aggregation was not formed when the formulation was placed under stress through agitation.

The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, Highly stabilized York, Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W. H. Freeman Pub., Highly stabilized York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W. H. Freeman Pub., Highly stabilized York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

Although the present invention is described primarily with reference to specific embodiments, it is also envisioned that other embodiments will become apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be contained within the present inventive methods.

EXAMPLES Example 1 Storage Stability of Aqueous Rfurin Formulations

The stability of a control furin formulation was tested at three different temperatures: room temperature (e.g., 25° C.), 37° C., and 45° C. These studies at elevated temperatures were used to study accelerated stability, which is comparable to long term studies of stability at lower temperatures. The samples were tested at time zero, then after 1, 2, 3, 4, and 7 days of incubation. Analyses included the furin activity assay, SEC, and western blotting.

Briefly, several 5 mL polypropylene vials were filled with 3 mL of rfurin and incubated at three different temperatures: ambient (room), 37° C. and 45° C. One vial from each incubation condition was taken out at time zero, then after 1, 2, 3, 4, and 7 days. 200 μL aliquots were made into 0.65 mL polypropylene vials and frozen at −80° C. awaiting analyses.

The stability of several furin formulations was determined under these conditions. Specifics of the various formulations are given in Table 17.

TABLE 17 Furin formulations tested for stability during storage at elevated temperatures. Control Formulation 10 mM Acetate 230 mM sodium chloride 1 mM calcium chloride pH 6.0 Test Formulation #1 46 mM HEPES 45 mM Acetate 190 mM sodium chloride 0.92 mM calcium chloride 75 ppm polysorbate 80 10% (w/w) trehalose dehydrate pH 6.0 Test Formulation #2 46 mM HEPES 45 mM Acetate 190 mM sodium chloride 0.92 mM calcium chloride 10% (w/w) trehalose dehydrate pH 6.0 Test Formulation #3 46 mM HEPES 45 mM Acetate 190 mM sodium chloride 0.92 mM calcium chloride 10 ppm polysorbate 80 10% (w/w) trehalose dehydrate pH 6.0 Test Formulation #4 46 mM HEPES 45 mM Acetate 190 mM sodium chloride 0.92 mM calcium chloride 25 ppm polysorbate 80 10% (w/w) trehalose dehydrate pH 6.0 Test Formulation #5 46 mM HEPES 45 mM Acetate 190 mM sodium chloride 0.92 mM calcium chloride 50 ppm polysorbate 80 10% (w/w) trehalose dehydrate pH 6.0 Test Formulation #6 46 mM HEPES 45 mM Acetate 190 mM sodium chloride 0.92 mM calcium chloride 100 ppm polysorbate 80 10% (w/w) trehalose dehydrate pH 6.0

One method of assessing stability is through a furin activity assay. In this assay, the activity of furin is determined from the rate of a substrate cleavage. The substrate, Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; AMC=7-amino-4-methoxy coumarin), is cleaved by furin and a fluorescent AMC is liberated. The assay is performed, for example, on an ELISA plate. The furin activity in a sample is determined from a standard curve created, for example, from a quality control rfurin standard. The activity values were calculated as mean values from two dilutions, each in duplicate.

rFurin stability at 37° C. in the highly stabilized formulation. The purpose of this experiment was to test rfurin stability in a stabilized formulation containing 75 ppm polysorbate 80. There was a small change in the composition of this sample because trehalose dihydrate (C₁₂H₂₂O₁₁*2H₂O, FW 378.33) was used, rather than, nonhydrated trehalose (C₁₂H₂₂O₁₁, FW 342.30). Use of the trehalose dihydrate created a slightly more dilute sample. Agitation studies suggested that the polysorbate 80 concentration should be kept between 50 ppm and 100 ppm to provide the best protection against rfurin denaturation induced by agitation. In this study, a rfurin sample in test formulation #1 was spiked with polysorbate 80 to 75 ppm and incubated at 37° C. for four days. An rfurin sample formulated with the control formulation served as a control.

Analyses by the Furin activity assay (FIG. 1) show loss of activity in the samples in the control formulation that were incubated at 37° C. and 45° C. Since the 45° C. sample was highly degraded and aggregated after just two days, the sample was not tested after that time. For the samples incubated at room temperature, the data was inconclusive as a result of the amount of variability in the assay, but the data suggested that the rfurin activity was relatively stable, with the activity after 7 days roughly equivalent to T=0.

In contrast to the data in the control formulation, analyses by the furin activity assay (FIG. 46) show that rfurin in test formulation #1 retained more of its activity than in the control formulation. After four days of incubation at 37° C., rfurin retained 76.7% of its original activity, while the control formulation preserved only 55.5% of its original activity (FIG. 47).

Size exclusion chromatograph (SEC) was also used to assess the stability of various furin formulations. For SEC methods, the following equipment and materials were used:

-   -   Agilent HPLC 1100 Series equipped with a temperature controlled         autosampler and a Photo-Diode Array detector     -   Autosampler 100 μL polypropylene vials     -   Size Exclusion Chromatography column from TOSOH Bioscience,         TSKgel     -   G3000SW×1, 7.8 mm ID×30 cm, 5 μm         In addition, following conditions were used for the SEC method:     -   Mobile Phase: 50 mM MOPS, 200 mM sodium sulfate, 0.02% sodium         azide, pH 7.0     -   Flow Rate: 0.5 mL/min     -   Run Time: 30 min     -   Sample Volume: 100 μL     -   Autosampler Temperature: 4° C.     -   Column Temperature: Ambient

Single wavelength absorption at 280 nm (Reference off) was used to create chromatographs. After manual integration, the height of the main peak at 19 min, corresponding to the rfurin content of the formulation, was used to evaluate stability. The reason for using the peak height instead of the peak area was that the chromatographic peaks were often not baseline separated, which could lead to large errors. Only one injection of each sample was conducted. Repeated analyses of the rfurin control formulation showed less than 2% variation in the peak height (data not shown).

The SEC results correlated with the furin activity assay results reported above. Analyses by SEC (FIG. 2) show that the height of the rfurin monomer peak (retention time 19 min) of the control formulation decreased with the time of incubation for all temperatures. The rate of the rfurin monomer peak decrease was relatively low in the samples incubated at room temperature, as 82% of the peak height remained after seven days of incubation. However, at 37° C., the height of the rfurin monomer peak dropped to 21.6%, of the original height, after seven days of incubation. At 45° C., after two days of incubation, the main peak decreased to 14.1%. Consistent with the loss of rfurin monomer content, other chromatographic peaks began to appear over time (FIG. 3), corresponding to aggregates (eluting at earlier time points) and degradation products (eluting at later time points). These additional peaks indicate both degradation and aggregation of rfurin in the control formulation. Increased degradation at 37° C. compared to room temperature storage was also confirmed by Western Blotting (FIG. 4). rFurin degradation may be caused by proteases that were co-purified with rfurin, or by the autocatalytic activity of rfurin itself.

In contrast, SEC analysis confirmed the increased stability of test formulation #1 (FIG. 48), containing trehalose, as compared to the control formulation, which does not contain a sugar or sugar alcohol. FIG. 48 shows that test formulation #1 maintained 74.8% of rfurin monomer peak height of the starting solution after incubation at 37° C. for 4 days, as compared to the control formulation, which maintained only 58.5% of rfurin monomer peak height of the starting solution during the same incubation.

Example 2 Storage Stability of Aqueous Rfurin Formulations Upon Freeze/Thaw

The stability of the control formulation and test formulations #2-6 were tested by a freeze/thaw study. Five freeze/thaw cycles were completed and samples were analyzed by the furin activity assay and SEC. Investigating the impact of freeze/thaw is important because rfurin samples in this study and rfurin BDS are stored frozen. Freeze/thaw experiments using the control formulation (n=4) demonstrate a minor loss of furin activity (approximately 7%) after a total of five freeze/thaw cycles. While there appears to be a downward trend, this may be due to true loss of protein or a reflection of assay variability. Regardless, this data supports the ability to freeze and thaw the rFurin, not only once, but multiple times, with little to no loss of activity. Likewise, SEC analysis of the oligomeric state of furin in the control formulation showed no loss in monomeric content over the course of the freeze/thaw experiment (FIG. 6).

Since rFurin is generally stored frozen, the impact of a thawing method was evaluated. Analyses by the activity assay (Figure) demonstrate no significant difference between the two thawing methods: thawing at 4° C. overnight, or at room temperature for 2 hours.

Taken together, these data suggest that rfurin in the control formulation is relatively stable for short periods at room temperature. Furthermore, loss of activity upon storage at elevated temperatures correlates to both rfurin degradation and aggregation. In addition, rfurin in the control formulation retained its activity well even after five freeze/thaw cycles.

Freeze/Thaw Study of rFurin in a highly stabilized Formulation. The freeze/thaw study was carried out using the same set of samples as the previous Freeze/thaw study; the stability of rfurin in test formulations #2 (0 ppm polysorbate 80), #3 (10 ppm polysorbate 80), #4 (25 ppm polysorbate 80), #5 (50 ppm polysorbate 80), and #6 (100 ppm polysorbate 80) was evaluated. Furin in the control formulation served as a control. Consistent with the prior analysis of the control formulation, analysis of furin activity (FIG. 43) demonstrated no significant loss of activity after five freeze/thaw cycles in any of the tested formulations. Analyses of the rfurin monomer content by SEC (FIGS. 44 and 45) demonstrated only a small trend of sample deterioration after freeze/thaw. Even after five freeze/thaw cycles, all samples retained more than 95% of their original activity.

Example 3 Storage Stability of Aqueous Rfurin Formulations Upon Mechanical Stress

Without being limited by theory, one potential mechanism of the agitation effect on rfurin samples is that it induces denaturation of rfurin, exposing hydrophobic surfaces. The hydrophobic surfaces are attracted to other hydrophobic surfaces, such as other denatured rFurin proteins which induces aggregation, or causing adsorption to hydrophobic surfaces on sample tubes and materials.

The agitation study was not designed to mimic the true process of rfurin use, but to overstress the sample. To be considered stabilized, a formulation of rfurin performs at least as well as the control formulation, and a highly stabilized formulation performs better than the control formulation.

The agitation study was carried out by shaking a rfurin sample in a bottle containing Teflon balls. Samples were taken after 2 and 17 hours of agitation and analyzed by furin activity assay and SEC. As shown in the results for both the furin activity assay and SEC analysis (FIGS. 7 and 8, respectively), rfurin in the control formulation is fairly resistant to agitation for a limited period of time. After two hours of vigorous agitation, such as might occur during a diafiltration step, more than 85% of rfurin was still active in the control formulation. However, after 17 hours of agitation, as might occur during shipping of a sample, less than 10% of the rfurin content remained active.

The effect was then tested on furin test formulations #2 to 6, which were created by spiking nine parts of a rFurin sample with one part of 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl2, pH 6 plus 10% w/w trehalose plus varied amounts of polysorbate 80.

This agitation study was carried out by shaking the test rfurin formulations and control formulation containing bottles with Teflon balls for three hours. Test formulations containing the following levels of polysorbate 80 were evaluated: none, 10 ppm, 25 ppm, 50 ppm, or 100 ppm. As shown in FIG. 31, polysorbate 80 in the test formulations provided significant protection to rfurin during agitation. After three hours of agitation, the activity of rFurin in the samples containing polysorbate 80 ranged from 31,499±1,218 U/mL to 36,787±847 U/mL, while the sample in the formulations without polysorbate 80 had 17,835±1,706 U/mL of Furin activity. The sample in the control formulation (10 mM sodium acetate, 230 mM sodium chloride, 1 mM calcium chloride, pH 6.0) had an activity of 24,368±1,135 U/mL.

Analyses by SEC confirmed the beneficial effect of polysorbate 80 in this study (FIG. 32). After three hours of agitation, the peak heights of rfurin in the samples containing polysorbate 80 ranged from 37.5 mAU to 35.9 mAU, while the height of the rFurin peak in the formulation without polysorbate 80 was 22.1 mAU. The height of the rFurin peak in the control formulation was 29.9 mAU. The effect of polysorbate 80 is also shown in FIGS. 33 and 34. The small trend suggests that the addition of 10 ppm and 25 ppm polysorbate 80, while still beneficial, is not as effective as use of 50 ppm or 100 ppm polysorbate 80. The SEC profile for the agitated sample in the control formulation (FIG. 35) does not show any additional peaks compared to the non-agitated sample. This would suggest that surface adsorption was the main reason for the activity loss. However, the UV absorption spectra (FIG. 36), show that the agitated sample has a slanted and elevated profile, which is a sign of light scattering and obscuration caused by the presence of aggregates. Light obscuration is generally caused by vary large aggregates that are more opaque than smaller molecules, which cause an elevation of the UV spectra. This phenomenon cannot be distinguished from absorption by a spectrophotometer because its detector measures the amount of light that passes through a sample. Since less light gets to the detector, it is reported as absorption; hence the absorption profile is elevated.

Light can also be scattered by aggregates, which would also decrease the amount of light getting into a detector. The shorter the wave length, the more light is scattered (the amount of scattered light is inversely proportional to the fourth power on the wavelength). This means that the shorter the wavelength the less light gets to a detector; hence the absorption profile is slanted. It is likely that the aggregates were present in this sample, but the SEC analysis was unable to detect them. These kinds of problems with SEC analyses are not uncommon as protein aggregates are often too large to pass through the column, or are absorbed by the column matrix. The UV absorption spectrum of the rfurin sample in the formulation without polysorbate 80 (FIG. 37) shows a similar elevated profile. Again, no aggregates were detected in the SEC analysis of this sample (FIG. 38). The samples formulated with polysorbate 80 show less aggregation than those without polysorbate 80 when analyzed by the UV absorption spectra (FIGS. 39 to 42). Although a UV spectrum cannot quantify the amount of aggregates, again, a small trend can be observed hinting that the samples with polysorbate 80 at 50 ppm and 100 ppm were better in protecting rFurin from aggregation than 10 ppm or 25 ppm. The data suggest that polysorbate 80 included in the formulation at a level of 75 ppm would be effective, as this concentration is in the middle of a wide plateau (50-10 ppm) and would represent a robust condition.

The UV spectrophotometry analysis was conducted using the following equipment and materials:

-   -   Agilent 8453 Spectrophotometer     -   Quartz cuvette 1 cm light pathway

The spectrophotometry analysis was conducted by first placing 0.5 mL of an appropriate formulation in a cuvette and measured as a blank. Then, 0.5 mL of a rfurin sample was placed in a cuvette, and the scan was taken from 240 nm to 400 nm.

Example 4 Impact of pH, Sucrose and Polysorbate 80 on rFurin Stability

rFurin samples were spiked with various buffers in order to adjust the pH to 5.0, 5.5, 6.0, 7.0 and 8.0. In addition, sucrose and/or polysorbate 80 were added to the samples at pH 6.0, 7.0 and 8.0. The samples were incubated at 37° C. and tested by the furin activity assay and SEC.

Both the furin activity assay (FIG. 11) and SEC (FIG. 12) showed that at 37° C. the rfurin was more stable at pH 6.0 than at pH 5.0 or 5.5. The samples at pH 6.0, in both the control formulation (FIG. 13) and in MES (FIG. 14), were more stable than those formulated in pH 7.0 or 8.0 (FIGS. 15 and 16) when analyzed by SEC. The addition of sucrose to the samples in pH 6.0 and 7.0 had a beneficial effect on rfurin stability. The SEC analyses (FIG. 17) showed that after four days of incubation at 37° C., the peak heights of the samples containing sucrose were on average at 75% of T=0, as compared to 52% without the sucrose. Analyses by the furin activity assay confirmed the results of the SEC experiments (FIGS. 18 and 19). The addition of sucrose improved rfurin stability in both the control formulation and in MES pH 6.0 (FIGS. 18 and 19), with similar stability seen using either buffer (FIG. 20). The effects of polysorbate 80 on rfurin stability in a sucrose-containing formulation at 37° C. were minimal, if any, as shown in FIGS. 17, 18, and 19.

These data suggest that at 37° C. rfurin is most stable in the pH 6.0 formulations compared to pH 5.0, 5.5, 7.0 and 8.0. The data further show that sucrose had a beneficial effect on rfurin stability at 37° C. In addition, no significant difference was seen in rfurin stability between the control buffer (acetate) or MES at pH 6.0 in the presence of 10% sucrose. Finally, there was no significant effect of polysorbate 80 on rfurin stability in a sucrose formulation at 37° C.

Example 5 Comparison of Furin Formulation Stability in Sucrose and Mannitol

As seen in Example 4, the addition of sucrose slowed down rfurin degradation and/or activity loss at 37° C. While sucrose is not used in downstream applications such as the rVWF process, other sugars and/or sugar alcohols are, such as mannitol. The addition of mannitol to the rfurin formulation, rather than sucrose, would therefore not introduce a new raw material to the vWF process. This study was conducted to determine whether the addition of mannitol would have a beneficial effect on rfurin stability. The test formulation also possessed an improved buffering capacity by addition of HEPES and acetic acid to final concentrations of 50 mM each at pH 6.0. A stabilized formulation was created by spiking a rfurin sample with 10% of 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl₂, pH 6, and then, adding either sucrose, mannitol, and/or polysorbate 80. Analyses by both the furin activity assay (FIG. 21) and SEC (FIG. 22) showed that after four days of incubation at 37° C., mannitol and sucrose had similar stabilizing effects on rfurin formulations. Again, the effect of polysorbate 80 on these formulations was not significant. Although the activity data (FIG. 21) show that the sample containing mannitol and polysorbate 80 after 4 days of incubation at 37° C. was better than other samples, not only does this sample not follow the overall pattern, but also the SEC analyses (Figure) did not confirm it. It is likely that this result was an outlier.

Example 6 Comparison of Furin Formulation Stability in Sucrose and Trehalose

Various publications have shown that mannitol accelerates the denaturation of proteins during freezing, which raised some concerns about its inclusion in the rfurin formulation buffer. No such problems have been reported with trehalose, another sugar already part of the rVWF production process. This study was conducted to determine whether addition of trehalose would have a beneficial effect on rfurin stability. In addition, various concentrations of polysorbate 80 were evaluated. Analyses using the furin activity assay (FIGS. 23 and 24) showed that after 5 days of incubation at 37° C., the sample containing 10% sucrose retained 66.2%±2.9% of its original activity while the sample containing 10% trehalose retained 60.1%±4%. The difference between these samples is not significant. Both samples were significantly better in preserving rfurin activity than the control formulation, which retained only 37.5%±4.2% of its original activity.

Analyses by SEC (FIG. 25) confirmed the activity analyses. The rfurin peak for the sample in 10% sucrose retained 78.3% of its height compared to 75.1% for the sample in 10% trehalose and 44.2% for the control formulation. Also, analyses by western blotting (FIG. 26) showed the least degradation in the samples containing 10% sucrose or 10% trehalose. Note that the samples are presented as pairs, with day 0 followed by day 5 for each of the formulations.

Both the furin activity assay (FIG. 27) and SEC (FIG. 28) analyses show that 10% trehalose was better than 5% or 2% in preserving rfurin activity at 37° C. These findings were also confirmed by western blotting (FIG. 26). The impact of polysorbate 80 on rfurin stability at 37° C. was minimal according to the analyses by the furin activity assay and SEC (FIGS. 29 and 30, respectively).

SEC and furin activity assays were conducted as described above. Western blotting was performed using the following equipment and materials:

-   -   Goat anti-Furin (peptide) primary antibody (Cat#SC-12484) from         Santa Cruz Biotechnology, Inc.     -   Rabbit anti-goat IgG-HRP conjugate secondary antibody         (Cat#A5420) from Sigma     -   Pierce Metal Enhanced Substrate Kit containing DAB/Metal         Concentrate 10×,     -   Pierce (Cat#1856090) and Stable Peroxidase Buffer, Pierce         (Cat#185591)     -   4-20% Tris-Glycine gel (Cat#EC6025BOX) from Invitrogen     -   NuPAGE® Sample Reducing Agent (0.5M DTT, 10×, Cat#NP0004) from         Invitrogen     -   Non-Reducing Sample Buffer (4×, Cat#84788) from Thermo     -   PBS (Cat#P4417) from Sigma     -   Tween 20 (Cat#9480) from EMD Chemicals     -   BSA (Cat#A7906) from Sigma     -   Gel transfer device (iBLOT Cat#IB401001) from Invitrogen     -   PVDF (Immobilon-P, Cat#IPVH07850) transfer membrane from         Millipore

A polyclonal antibody was used in this study instead of a monoclonal to increase detection of rFurin degradation products. 5 μl of a sample was combined with 1 μl of 10×DTT, and 4 μl of 4× non-reducing dye. The sample was heated at 70° C. to 75° C. for 10 minutes. 10 μl of the sample was loaded to 4-20% SDS-PAGE gel, and run at 150 V for 60 to 70 minutes. The protein gel was transferred to PVDF membrane using the iBLOT at 20 V for 6 minutes. The PVDF membrane was then blocked with PBS buffer containing 0.1% TWEEN 20 and 3% BSA for 1 hour. The membrane was incubated for one hour in the primary antibody diluted 125 fold with the same buffer. The membrane was washed with PBST buffer for five minutes, three times each. The membrane was incubated for one hour in the secondary antibody diluted 10,000 fold with the same buffer as the primary antibody. The incubation time was 1 hour. The blot was washed using PBST buffer for five minutes, three times each. The blot was developed in 10 mL of premixed chromagenic substrate (1 ml concentrate plus 9 ml of Stable Peroxidase Buffer) followed by the wash with water for 10 minutes.

Example 7 Effects of Stabilizing Agents Upon Dilution of Furin Compositions

The rFurin dilution study was carried out to compare a starting formulation and its diluent to a stabilized formulation and a stabilized diluent. The study mimicked the dilution method that used in the rVWF maturation process. Briefly, rfurin BDS was passed through a filter followed by the addition of a 200-fold volume of the diluent. The main difference between the test dilution method and the starting dilution method is that in the test (highly stabilized) method, 75 ppm polysorbate 80 is present in both the rfurin BDS and the diluent, while in the starting method, there is no polysorbate 80 in either the rfurin BDS or the diluent.

Two dilution experiments were carried out, and both have shown that the combination of the stabilized formulation with its stabilized diluent was superior to the starting furin and diluent formulations. In the first experiment, the test method using the stabilized formulations recovered 83% to 87% of the expected rfurin activity, while the control methods recovered only 21% to 22% (Table 18). In this experiment, the rfurin sample was passed through a filter and followed by a 200-fold volume of a diluent. The filtrate was collected to a bag and then transferred to a graduated cylinder.

TABLE 18 Experimental results of rfurin dilution experiments performed with and without non-ionic surfactant. Activity St. Dilution Expected Recovery Sample (IU) Dev. Factor Activity (IU) (% Activity) Starting Material 25,953 1,118 1 N/A N/A Control Formulation From a Bag 27.264 2.815 200 129.8 21.0 After Filtration From a 28.3 1.56 200 129.8 21.8 Cylinder New Formulation From a Bag 108.137 6.611 200 129.8 83.3 After Filtration From a 113.4 5.21 200 129.8 87.4 Cylinder

In the second experiment, the test method recovered 80% of the expected rfurin activity, while the control method recovered only 27% (Table 19). In this experiment, the rfurin sample was passed through a filter and followed by a 200-fold volume of a diluent. The filtrate was collected to a cylinder. The combination of the stabilized rfurin formulation and the stabilized diluent increased rfurin recovery three to four times compared to the control method. The improved recovery is, without being limited by theory, linked to the presence of polysorbate 80.

TABLE 19 Experimental results of rfurin dilution experiments performed with and without non-ionic surfactant. Expected Recovery Sample Activity (IU) St. Dev. Dilution Factor Activity (IU) (% Activity) Starting Material 32,379 1,654 N/A N/A N/A Control Formulation 44.267 1.607 200 161.9 27.3 After Filtration New Formulation 129.646 11.514 200 161.9 80.1 After Filtration

Example 8 Characteristics of Stabilized Formulation Stock Buffer

The stock buffer for the stabilized rfurin formulation (500 mM HEPES, 400 mM acetic acid, 1 mM CaCl₂, pH 6.0) can be made consistently (Table 20). The RSD's for pH and conductivity were <0.3%. The pH was slightly and indirectly correlated with temperature, dropping 0.014 units per degree Celsius, while conductivity was directly correlated with temperature, raising 0.40 mS/cm per degree C. (FIG. 49). The average temperature of the samples when the pH and conductivity were measured was 27.6° C. In manufacturing, the samples are read at 25±0.5° C. Based on the linear regressions in FIG. 48, the expected pH and conductivity at 25° C. were calculated. The relative standard deviation from Table 20 was applied to this value to determine the expected range (±3 sd). pH at 25° C. is expected to be 6.04 (±0.01). Conductivity at 25° C. is expected to be 19.9±0.2 mS/cm. These expected ranges are based on three lots made under limited circumstances. The specification ranges for the buffer need not be set this strictly. The pH specification will be the same as the final BDS pH specification, 5.90-6.10. The conductivity range will be similar to that used for the previous BDS buffer, e.g., with a range of ±20%, for a specification of 15.9-23.9 mS/cm. The requirements of the rVWF process are generally not of concern in setting the buffer specifications because the rfurin will be diluted 200-fold in the rVWF process. Experimental data in Table 21 and Table 22 correspond to the graphs presented in FIG. 49.

TABLE 20 Characteristics of stabilized formulation stock buffer. Reagent Buffer 1 Buffer 2 Buffer 3 mean sd rsd, % mean + 3 sd mean − 3 sd Water, L 0.85 0.85 0.85 HEPES, g 119.15 119.15 119.15 CaCl2, g 0.111 0.111 0.111 Acetic acid, mL 23 23 23 10M NaOH, mL 39.4 39.4 39.4 pH 6.011 6.003 6.005 6.006 0.004 0.07 6.019 5.994 Cond., mS/cm 21.23 21.15 21.11 21.16 0.06 0.29 21.35 20.98 Temp., C. 27.5 27.6 27.6 27.6 0.06 0.21 27.7 27.4

TABLE 21 Dependence of stock buffer conductivity on temperature. Temp, C. Cond, mS/cm

18 17.23

18.4 17.42

18.7 17.53

19 17.63

19.3 17.75

19.6 17.86

19.7 17.91

20 18.03

20.2 17.72

20.5 17.83

21.6 17.86

24.4 19.81

27.6 21.1 

indicates data missing or illegible when filed

TABLE 22 Dependence of stock buffer pH on temperature. Temp, C. pH 18 6.135 19.3 6.103 21.6 6.088 27.6 6.005

REFERENCES

All references cited herein are hereby incorporated by reference herein in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled. 

1. A stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: (a) from 8,000 U/mL to 500,000 U/mL rfurin; (b) from 100 mM to 300 mM of a pharmaceutically acceptable salt; (c) from 0.5 mM to 2 mM calcium; (d) from 2% to 20% sugar or sugar alcohol; (e) from 10 to 200 ppm non-ionic surfactant; (f) from 10 to 200 mM buffering agent; and (g) a pH from 5.5 to 7.5.
 2. (canceled)
 3. The composition of claim 1, comprising 190±10 mM of a pharmaceutically acceptable salt.
 4. (canceled)
 5. The composition of claim 1, wherein the pharmaceutically acceptable salt is sodium chloride.
 6. (canceled)
 7. The composition of claim 1, comprising 0.9±0.2 mM calcium. 8-9. (canceled)
 10. The composition of claim 1, comprising 10±2% sugar or sugar alcohol.
 11. (canceled)
 12. The composition of claim 1, wherein the sugar or sugar alcohol is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof. 13-15. (canceled)
 16. The composition of claim 1, comprising from 50 ppm to 100 ppm non-ionic surfactant.
 17. (canceled)
 18. The composition of claim 1, wherein the non-ionic surfactant is polysorbate-80.
 19. (canceled)
 20. The composition of claim 1, comprising 90±10 mM buffering agent. 21-24. (canceled)
 25. The composition of claim 1, wherein the buffering agent comprises acetate and HEPES.
 26. (canceled)
 27. The composition of claim 25, comprising 45±5 mM acetate and 45±5 mM HEPES. 28-30. (canceled)
 31. The composition of claim 1, having a pH of 6.0±0.2. 32-41. (canceled)
 42. A stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: (a) from 8,000 U/mL to 500,000 U/mL rfurin; (b) 190 mM sodium chloride; (c) 0.92 mM calcium; (d) 10% trehalose; (e) 75 ppm polysorbate 80; (f) 45 mM acetic acid; (g) 46 mM HEPES; and (g) a pH from 5.5 to 7.5. 43-44. (canceled)
 45. The composition of claim 42, having a pH of 6.0±0.2.
 46. (canceled)
 47. A method for diluting an aqueous rfurin composition, the method comprising adding a dilution buffer comprising from 10 ppm to 200 ppm of a non-ionic surfactant to the aqueous rfurin composition, thereby forming a diluted rfurin composition. 48-56. (canceled)
 57. The composition of claim 3, comprising 0.9±0.2 mM calcium.
 58. The composition of claim 3, comprising 10±2% sugar or sugar alcohol.
 59. The composition of claim 7, comprising 10±2% sugar or sugar alcohol.
 60. The composition of claim 3, comprising from 50 ppm to 100 ppm non-ionic surfactant.
 61. The composition of claim 7, comprising from 50 ppm to 100 ppm non-ionic surfactant.
 62. The composition of claim 10, comprising from 50 ppm to 100 ppm non-ionic surfactant.
 63. The composition of claim 3, comprising 90±10 mM buffering agent.
 64. The composition of claim 7, comprising 90±10 mM buffering agent.
 65. The composition of claim 10, comprising 90±10 mM buffering agent.
 66. The composition of claim 16, comprising 90±10 mM buffering agent.
 67. The composition of claim 3, having a pH of 6.0±0.2.
 68. The composition of claim 7, having a pH of 6.0±0.2.
 69. The composition of claim 10, having a pH of 6.0±0.2.
 70. The composition of claim 16, having a pH of 6.0±0.2.
 71. The composition of claim 20, having a pH of 6.0±0.2. 